Lipoprotein complexes and manufacturing and uses thereof

ABSTRACT

The present disclosure relates to lipoprotein complexes and lipoprotein populations and their use in the treatment and/or prevention of dyslipidemic diseases, disorders, and/or conditions. The disclosure further relates to recombinant expression of apolipoproteins, purification of apolipoproteins, and production of lipoprotein complexes using thermal cycling-based methods.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 US.C. §119(e) ofprovisional application No. 61/440,371, filed Feb. 7, 2011, provisionalapplication No. 61/452,630, filed Mar. 14, 2011, and provisionalapplication No. 61/487,263, filed May 17, 2011, the contents of all ofwhich are incorporated herein in their entireties by reference thereto.

2. TECHNICAL FIELD

The present disclosure provides lipoprotein complexes, pharmaceuticalcompositions comprising the complexes, methods of producing andpurifying apolipoproteins for such complexes, methods of making thecomplexes and methods of using the complexes to treat or preventcardiovascular diseases, disorders, and/or conditions associatedtherewith.

3. BACKGROUND 3.1. Overview

Circulating cholesterol is carried by plasma lipoproteins—complexparticles of lipid and protein composition that transport lipids in theblood. Four major classes of lipoprotein particles circulate in plasmaand are involved in the fat-transport system: chylomicrons, very lowdensity lipoprotein (VLDL), low density lipoprotein (LDL) and highdensity lipoprotein (HDL). Chylomicrons constitute a short-lived productof intestinal fat absorption. VLDL and, particularly, LDL areresponsible for the delivery of cholesterol from the liver (where it issynthesized or obtained from dietary sources) to extrahepatic tissues,including the arterial walls. HDL, by contrast, mediates reversecholesterol transport (RCT), the removal of cholesterol lipids, inparticular from extrahepatic tissues to the liver, where it is stored,catabolized, eliminated or recycled. HDL also plays a beneficial role ininflammation, transporting oxidized lipids and interleukin, which may inturn reduce inflammation in blood vessel walls.

Lipoprotein particles have a hydrophobic core comprised of cholesterol(normally in the form of a cholesteryl ester) and triglycerides. Thecore is surrounded by a surface coat comprising phospholipids,unesterified cholesterol and apolipoproteins. Apolipoproteins mediatelipid transport, and some may interact with enzymes involved in lipidmetabolism. At least ten apolipoproteins have been identified,including: ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II,ApoC-III, ApoD, ApoE, ApoJ and ApoH. Other proteins such as LCAT(lecithin:cholesterol acyltransferase), CETP (cholesteryl ester transferprotein), PLTP (phospholipid transfer protein) and PON (paraoxonase) arealso found associated with lipoproteins.

Cardiovascular diseases such as coronary heart disease, coronary arterydisease and atherosclerosis are linked overwhelmingly to elevated serumcholesterol levels. For example, atherosclerosis is a slowly progressivedisease characterized by the accumulation of cholesterol within thearterial wall. Compelling evidence supports the theory that lipidsdeposited in atherosclerotic lesions are derived primarily from plasmaLDLs; thus, LDLs have popularly become known as “bad” cholesterol. Incontrast, HDL serum levels correlate inversely with coronary heartdisease. Indeed, high serum levels of HDLs are regarded as a negativerisk factor. It is hypothesized that high levels of plasma HDLs are notonly protective against coronary artery disease, but may actually induceregression of atherosclerotic plaque (see, e.g., Badimon et al., 1992,Circulation 86 (Suppl. III):86-94; Dansky and Fisher, 1999, Circulation100:1762-63; Tangirala et al., 1999, Circulation 100(17):1816-22; Fan etal., 1999, Atherosclerosis 147(1):139-45; Deckert et al., 1999,Circulation 100(11):1230-35; Boisvert et al., 1999, Arterioscler.Thromb. Vasc. Biol. 19(3):525-30; Benoit et al., 1999, Circulation99(1):105-10; Holvoet et al., 1998, J. Clin. Invest. 102(2):379-85;Duverger et al., 1996, Circulation 94(4):713-17; Miyazaki et al., 1995,Arterioscler. Thromb. Vasc. Biol. 15(11):1882-88; Mezdour et al., 1995,Atherosclerosis 113(2):237-46; Liu et al., 1994, J. Lipid Res.35(12):2263-67; Plump et al., 1994, Proc. Nat. Acad. Sci. USA91(20):9607-11; Paszty et al., 1994, J. Clin. Invest. 94(2):899-903; Sheet al, 1992, Chin. Med. J. (Engl). 105(5):369-73; Rubin et al., 1991,Nature 353(6341):265-67; She et al., 1990, Ann. NY Acad. Sci.598:339-51; Ran, 1989, Chung Hua Ping Li Hsueh Tsa Chih (also translatedas: Zhonghua Bing Li Xue Za Zhi) 18(4):257-61; Quezado et al., 1995, J.Pharmacol. Exp. Ther. 272(2).604-11; Duverger et al., 1996,Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29; Kopfler et al., 1994,Circulation; 90(3):1319-27; Miller et al., 1985, Nature314(6006):109-11; Ha et al., 1992, Biochim. Biophys. Acta1125(2):223-29; Beitz et al., 1992, Prostaglandins Leukot. Essent. FattyAcids 47(2):149-52). As a consequence, HDLs have popularly become knownas “good” cholesterol, (see, e.g., Zhang, et al., 2003 Circulation108:661-663).

The “protective” role of HDL has been confirmed in a number of studies(e.g., Miller et al., 1977, Lancet 1(8019):965-68; Whayne et al., 1981,Atherosclerosis 39:411-19). In these studies, the elevated levels of LDLappear to be associated with increased cardiovascular risk, whereas highHDL levels seem to confer cardiovascular protection. In vivo studieshave further demonstrated the protective role of HDL, showing that HDLinfusions into rabbits may hinder the development of cholesterol inducedarterial lesions (Badimon et al., 1989, Lab. Invest. 60:455-61) and/orinduce their regression (Badimon et al., 1990, J. Clin. Invest.85:1234-41).

3.2. Reverse Cholesterol Transport, HDL And Apolipoprotein A-I

The reverse cholesterol transport (RCT) pathway functions to eliminatecholesterol from most extrahepatic tissues and is crucial to maintainingthe structure and function of most cells in the body. RCT consistsmainly of three steps: (a) cholesterol efflux, i.e., the initial removalof cholesterol from various pools of peripheral cells; (b) cholesterolesterification by the action of lecithin:cholesterol acyltransferase(LCAT), preventing a re-entry of effluxed cholesterol into cells; and(c) uptake of HDL-cholesterol and cholesteryl esters to liver cells forhydrolysis, then recycling, storage, excretion in bile or catabolism tobile acids.

LCAT, the key enzyme in RCT, is produced by the liver and circulates inplasma associated with the HDL fraction. LCAT converts cell-derivedcholesterol to cholesteryl esters, which are sequestered in HDL destinedfor removal (see Jonas 2000, Biochim. Biophys. Acta 1529(1-3):245-56).Cholesteryl ester transfer protein CETP) and phospholipid transferprotein (PLTP) contribute to further remodeling of the circulating HDLpopulation. CETP moves cholesteryl esters made by LCAT to otherlipoproteins, particularly ApoB-comprising lipoproteins, such as VLDLand LDL. PLTP supplies lecithin to HDL. HDL triglycerides arecatabolized by the extracellular hepatic triglyceride lipase, andlipoprotein cholesterol is removed by the liver via several mechanisms.

The functional characteristics of HDL particles are mainly determined bytheir major apolipoprotein components such as ApoA-I and ApoA-II. Minoramounts of ApoC-I, ApoC-II, ApoC-III, ApoD, ApoA-IV, ApoE, and ApoJ havealso been observed associated with HDL. HDL exists in a wide variety ofdifferent sizes and different mixtures of the above-mentionedconstituents, depending on the status of remodeling during the metabolicRCT cascade or pathway.

Each HDL particle usually comprises at least 1 molecule, and usually twoto 4 molecules, of ApoA-I. HDL particles may also comprise only ApoE(gamma-LpE particles), which are known to also be responsible forcholesterol efflux, as described by Prof. Gerd Assmann (see, e.g., vonEckardstein et al., 1994, Curr Opin Lipidol. 5(6):404-16). ApoA-I issynthesized by the liver and small intestine as preproApolipoproteinA-I, which is secreted as proApolipoprotein A-I (proApoA-I) and rapidlycleaved to generate the plasma form of ApoA-I, a single polypeptidechain of 243 amino acids (Brewer et al., 1978, Biochem. Biophys. Res.Commun. 80:623-30). PreproApoA-I that is injected experimentallydirectly into the bloodstream is also cleaved into the plasma form ofApoA-I (Klon et al., 2000, Biophys. J. 79(3):1679-85; Segrest et al.,2000, Curr. Opin. Lipidol. 11(2):105-15; Segrest et al., 1999, J. Biol.Chem. 274 (45):31755-58).

ApoA-I comprises 6 to 8 different 22-amino acid alpha-helices orfunctional repeats spaced by a linker moiety that is frequently proline.The repeat units exist in amphipathic helical conformation (Segrest etal., 1974, FEBS Lett. 38: 247-53) and confer the main biologicalactivities of ApoA-I, i.e., lipid binding and lecithin cholesterol acyltransferase (LCAT) activation.

ApoA-I forms three types of stable complexes with lipids: small,lipid-poor complexes referred to as pre-beta-1 HDL; flattened discoidalparticles comprising polar lipids (phospholipid and cholesterol)referred to as pre-beta-2 HDL; and spherical particles, comprising bothpolar and nonpolar lipids, referred to as spherical or mature EIDL (HDL₃and HDL₂). Most HDL in the circulating population comprises both ApoA-Iand ApoA-H (the “AI/AII-IIDL fraction”). However, the fraction of HDLcomprising only ApoA-I (the “AI-HDL fraction”) appears to be moreeffective in RCT. Certain epidemiologic studies support the hypothesisthat the ApoA-I-HDL fraction is anti-atherogenic (Parra et al., 1992,Arterioscler. Thromb. 12:701-07; Decossin et al., 1997, Eur. J. Clin.Invest. 27:299-307).

HDL particles are made of several populations of particles that havedifferent sizes, lipid composition and apolipoprotein composition. Theycan be separated according to their properties, including their hydrateddensity, apolipoprotein composition and charge characteristics. Forexample, the pre-beta-HDL fraction is characterized by a lower surfacecharge than mature alpha-HDL. Because of this charge difference,pre-beta-HDL and mature alpha-HDL have different electrophoreticmobilities in agarose gel (David et al., 1994, J. Biol. Chem.269(12):8959-8965).

The metabolism of pre-beta-HDL and mature alpha-HDL also differs.Pre-beta-HDL has two metabolic fates: either removal from plasma andcatabolism by the kidney or remodeling to medium-sized HDL that arepreferentially degraded by the liver (Lee et al., 2004, J. Lipid Res.45(4):716-728).

Although the mechanism for cholesterol transfer from the cell surface(i.e., cholesterol efflux) is unknown, it is believed that thelipid-poor complex, pre-beta-1 HDL, is the preferred acceptor forcholesterol transferred from peripheral tissue involved in RCT (seeDavidson et al., 1994, J. Biol. Chem. 269:22975-82; Bielicki et al.,1992, J. Lipid Res. 33:1699-1709; Rothblat et al., 1992, J. Lipid Res.33:1091-97; and Kawano et al., 1993, Biochemistry 32:5025-28; Kawano etal., 1997, Biochemistry 36:9816-25). During this process of cholesterolrecruitment from the cell surface, pre-beta-1 HDL is rapidly convertedto pre-beta-2 HDL. PLTP may increase the rate of pre-beta-2 HDL discformation, but data indicating a role for PLTP in RCT are lacking. LCATreacts preferentially with discoidal, small (pre-beta) and spherical(i.e., mature) HDL, transferring the 2-acyl group of lecithin or otherphospholipids to the free hydroxyl residue of cholesterol to generatecholesteryl esters (retained in the HDL) and lysolecithin. The LCATreaction requires ApoA-I as an activator; i.e., ApoA-I is the naturalcofactor for LCAT. The conversion of cholesterol sequestered in the HDLto its ester prevents re-entry of cholesterol into the cell, the netresult being that cholesterol is removed from the cell.

Cholesteryl esters in the mature HDL particles in the ApoAI-HDL fraction(i.e., comprising ApoA-I and no ApoA-II) are removed by the liver andprocessed into bile more effectively than those derived from HDLcomprising both ApoA-I and ApoA-II (the Al/AII-HDL fraction). This maybe owed, in part, to the more effective binding of ApoAI-HDL to thehepatocyte membrane. The existence of an HDL receptor has beenhypothesized, and a scavenger receptor, class B, type I (SR-BI) has beenidentified as an HDL receptor (Acton et al., 1996, Science 271:518-20;Xu et al., 1997, Lipid Res. 38:1289-98). SR-BI is expressed mostabundantly in steroidogenic tissues (e.g., the adrenals), and in theliver (Landschulz et al., 1996, J. Clin. Invest. 98:984-95; Rigotti etal., 1996, J. Biol. Chem. 271:33545-49). For a review of HDL receptors,see Broutin et al., 1988, Anal. Biol. Chem. 46:16-23.

Initial lipidation by ATP-binding cassette transporter AI appears to becritical for plasma HDL formation and for the ability of pre-beta-HDLparticles to effect cholesterol efflux (Lee and Parks, 2005, Curr. Opin.Lipidol. 16(1):19-25). According to these authors, this initiallipidation enables pre-beta-HDL to function more efficiently as acholesterol acceptor and prevents ApoA-I from rapidly associating withpre-existing plasma HDL particles, resulting in greater availability ofpre-beta-HDL particles for cholesterol efflux.

CETP may also play a role in RCT. Changes in CETP activity or itsacceptors, VLDL and LDL, play a role in “remodeling” the HDL population.For example, in the absence of CETP, the HDLs become enlarged particlesthat are not cleared. (For reviews of RCT and HDLs, see Fielding andFielding, 1995, J. Lipid Res. 36:211-28; Barrans et al., 1996, Biochem.Biophys. Acta 1300:73-85; Hirano et al., 1997, Arterioscler. Thromb.Vasc. Biol. 17(6):1053-59).

HDL also plays a role in the reverse transport of other lipids andapolar molecules, and in detoxification, i.e., the transport of lipidsfrom cells, organs, and tissues to the liver for catabolism andexcretion. Such lipids include sphingomyelin (SM), oxidized lipids, andlysophophatidylcholine. For example, Robins and Fasulo (1997, J. Clin.Invest. 99:380-84) have shown that HDLs stimulate the transport of plantsterol by the liver into bile secretions.

The major component of HDL, ApoA-I, can associate with SM in vitro. WhenApoA-I is reconstituted in vitro with bovine brain SM (BBSM), a maximumrate of reconstitution occurs at 28° C., the temperature approximatingthe phase transition temperature for BBSM (Swaney, 1983, J. Biol. Chem.258(2), 1254-59). At BBSM:ApoA-I ratios of 7.5:1 or less (wt/wt), asingle reconstituted homogeneous HDL particle is formed that comprisesthree ApoA-I molecules per particle and that has a BBSM:ApoA-I molarratio of 360:1. It appears in the electron microscope as a discoidalcomplex similar to that obtained by recombination of ApoA-I withphosphatidylcholine at elevated ratios of phospholipid:protein. AtBBSM:ApoA-I ratios of 15:1 (wt/wt), however, larger-diameter discoidalcomplexes form that have a higher phospholipid:protein molar ratio(535:1). These complexes are significantly larger, more stable, and moreresistant to denaturation than ApoA-I complexes formed withphosphatidylcholine.

Sphingomyelin (SM) is elevated in early cholesterol acceptors(pre-beta-HDL and gamma-migrating ApoE-comprising lipoprotein),suggesting that SM might enhance the ability of these particles topromote cholesterol efflux (Dass and Jessup, 2000, J. Pharm. Pharmacol.52:731-61; Huang et al., 1994, Proc. Natl. Acad. Sci. USA 91:1834-38;Fielding and Fielding 1995, J. Lipid Res. 36:211-28).

3.3. Protective Mechanism of HDL and ApoA-I

Studies of the protective mechanism(s) of HDL have focused onApolipoprotein A-I (ApoA-I), the major component of HDL. High plasmalevels of ApoA-I are associated with absence or reduction of coronarylesions (Maciejko et al., 1983, N. Engl. J. Med. 309:385-89; Sedlis etal., 1986, Circulation 73:978-84).

The infusion of ApoA-I or of HDL in experimental animals exertssignificant biochemical changes, as well as reduces the extent andseverity of atherosclerotic lesions. After an initial report by Maciejkoand Mao (1982, Arteriosclerosis 2:407a), Badimon et al., (1989, Lab.Invest. 60:455-61; 1989, J. Clin. Invest. 85:1234-41) found that theycould significantly reduce the extent of atherosclerotic lesions(reduction of 45%) and their cholesterol ester content (reduction of58.5%) in cholesterol-fed rabbits, by infusing HDL (d=1.063-1.325 g/ml).They also found that the infusions of HDL led to a close to a 50%regression of established lesions. Esper et al. (1987, Arteriosclerosis7:523a) have shown that infusions of HDL can markedly change the plasmalipoprotein composition of Watanabe rabbits with inheritedhypercholesterolemia, which develop early arterial lesions. In theserabbits, HDL infusions can more than double the ratio between theprotective HDL and the atherogenic LDL.

The potential of HDL to prevent arterial disease in animal models hasbeen further underscored by the observation that ApoA-I can exert afibrinolytic activity in vitro (Saku et al., 1985, Thromb. Res. 39:1-8).Ronneberger (1987, Xth Int. Congr. Pharmacol., Sydney, 990) demonstratedthat ApoA-I can increase fibrinolysis in beagle dogs and in Cynomologousmonkeys. A similar activity can be noted in vitro on human plasma.Ronneberger was able to confirm a reduction of lipid deposition andarterial plaque formation in ApoA-I treated animals.

In vitro studies indicate that complexes of ApoA-I and lecithin canpromote the efflux of free cholesterol from cultured arterial smoothmuscle cells (Stein et al., 1975, Biochem. Biophys. Acta, 380:106-18).By this mechanism, HDL can also reduce the proliferation of these cells(Yoshida et al., 1984, Exp. Mol Pathol. 41:258-66).

Infusion therapy with HDL comprising ApoA-I or ApoA-I mimetic peptideshas also been shown to regulate plasma HDL levels by the ABC1transporter, leading to efficacy in the treatment of cardiovasculardisease (see, e.g., Brewer et al., 2004, Arterioscler. Thromb. Vasc.Biol. 24:1755-1760).

Two naturally occurring human polymorphism of ApoA-I have been isolatedin which an arginine residue is substituted with cysteine. InApolipoprotein A-I_(Milano) (ApoA-I_(M)), this substitution occurs atresidue 173, whereas in Apolipoprotein A-I_(Paris) (ApoA-Ip), thissubstitution occurs at residue 151 (Franceschini et al., 1980, J. Clin.Invest. 66:892-900; Weisgraber et al., 1983, J. Biol. Chem. 258:2508-13;Bruckert et al., 1997, Atherosclerosis 128:121-28; Daum et al., 1999, J.Mol. Med. 77:614-22; Klon et al., 2000, Biophys. J. 79(3):1679-85). Yeta further naturally occurring human polymorphism of ApoA-I has beenisolated, in which a leucine is substituted with an arginine at position144. This polymorphism has been termed Apolipoprotein A-I Zaragoza(ApoA-I_(Z)) and is assocated with severe hypoalphalipoproteinemia andan enhanced effect of high density lipoprotein (HDL) reverse cholesteroltransport (Recalde et al., 2001, Atherosclerosis 154(3):613-623;Fiddyment et al., 2011, Protein Expr. Purif. 80(1):110-116).

Reconstituted HDL particles comprising disulfide-linked homodimers ofeither ApoA-I_(M) or ApoA-I_(P) are similar to reconstituted HDLparticles comprising wild-type ApoA-I in their ability to cleardimyristoylphosphatidylcholine (DMPC) emulsions and their ability topromote cholesterol efflux (Calabresi et al., 1997b, Biochemistry36:12428-33; Franceschini et al., 1999, Arterioscler. Thromb. Vasc.Biol. 19:1257-62; Daum et al., 1999, J. Mol. Med. 77:614-22). In bothmutations, heterozygous individuals have decreased levels of HDL butparadoxically, are at a reduced risk for atherosclerosis (Franceschiniet al., 1980, J. Clin. Invest. 66:892-900; Weisgraber et al., 1983, J.Biol. Chem. 258:2508-13; Bruckert et al., 1997, Atherosclerosis128:121-28). Reconstituted HDL particles comprising either variant arecapable of LCAT activation, although with decreased efficiency whencompared with reconstituted HDL particles comprising wild-type ApoA-I(Calabresi et al., 1997, Biochem. Biophys. Res. Commun. 232:345-49; Daumet al., 1999, J. Mol. Med. 77:614-22).

The ApoA-I_(M) mutation is transmitted as an autosomal dominant trait;eight generations of carriers within a family have been identified(Gualandri et al., 1984, Am. J. Hum. Genet. 37:1083-97). The status ofan ApoA-I_(M) carrier individual is characterized by a remarkablereduction in HDL-cholesterol level. In spite of this, carrierindividuals do not apparently show any increased risk of arterialdisease. Indeed, by examination of genealogical records, it appears thatthese subjects may be “protected” from atherosclerosis (Sirtori et al.,2001, Circulation, 103: 1949-1954; Roma et al., 1993, J. Clin. Invest.91(4):1445-520).

The mechanism of the possible protective effect of ApoA-I_(M) incarriers of the mutation seems to be linked to a modification in thestructure of the mutant ApoA-I_(M), with loss of one alpha-helix and anincreased exposure of hydrophobic residues (Franceschini et al., 1985,J. Biol. Chem. 260:1632-35). The loss of the tight structure of themultiple alpha-helices leads to an increased flexibility of themolecule, which associates more readily with lipids, compared to normalApoA-I. Moreover, lipoprotein complexes are more susceptible todenaturation, thus suggesting that lipid delivery is also improved inthe case of the mutant.

Bielicki, et al. (1997, Arterioscler. Thromb. Vasc. Biol. 17(9):1637-43) has demonstrated that ApoA-I_(M) has a limited capacity torecruit membrane cholesterol compared with wild-type ApoA-I. Inaddition, nascent HDL formed by the association of ApoA-I_(M) withmembrane lipids was predominantly 7.4-nm particles rather than larger 9-and 11-nm complexes formed by wild-type ApoA-I. These observationsindicate that the Arg₁₇₃→Cys₁₇₃ substitution in the ApoA-I primarysequence interfered with the normal process of cellular cholesterolrecruitment and nascent HDL assembly. The mutation is apparentlyassociated with a decreased efficiency for cholesterol removal fromcells. Its antiatherogenic properties may therefore be unrelated to RCT.

The most striking structural change attributed to the Arg₁₇₃→Cys₁₇₃substitution is the dimerization of ApoA-I_(M) (Bielicki et al., 1997,Arterioscler. Thromb. Vasc. Biol. 17 (9):1637-43). ApoA-I_(M) can formhomodimers with itself and heterodimers with ApoA-II. Studies of bloodfractions comprising a mixture of apolipoproteins indicate that thepresence of dimers and complexes in the circulation may be responsiblefor an increased elimination half-life of apolipoproteins. Such anincreased elimination half-life has been observed in clinical studies ofcarriers of the mutation (Gregg et al., 1988, NATO ARW on HumanApolipoprotein Mutants: From Gene Structure to Phenotypic Expression,Limone S G). Other studies indicate that ApoA-I_(M) dimers(ApoA-I_(M)/ApoA-I_(M)) act as an inhibiting factor in theinterconversion of HDL particles in vitro (Franceschini et al., 1990, J.Biol. Chem. 265:12224-31).

3.4. Current Treatments for Dyslipidemia and Related Disorders

Dyslipidemic disorders are diseases associated with elevated serumcholesterol and triglyceride levels and lowered serum HDL:LDL ratios,and include hyperlipidemia, especially hypercholesterolemia, coronaryheart disease, coronary artery disease, vascular and perivasculardiseases, and cardiovascular diseases such as atherosclerosis. Syndromesassociated with atherosclerosis such as transient ischemic attack orintermittent claudication, caused by arterial insufficiency, are alsoincluded. A number of treatments are currently available for loweringthe elevated serum cholesterol and triglycerides associated withdyslipidemic disorders. However, each has its own drawbacks andlimitations in terms of efficacy, side-effects and qualifying patientpopulation.

Bile-acid-binding resins are a class of drugs that interrupt therecycling of bile acids from the intestine to the liver; e.g.,cholestyramine (Questran Light®, Bristol-Myers Squibb), colestipolhydrochloride (Colestid®, The Upjohn Company), and colesevelamhydrochloride (Welchol®, Daiichi-Sankyo Company). When taken orally,these positively-charged resins bind to the negatively charged bileacids in the intestine. Because the resins cannot be absorbed from theintestine, they are excreted carrying the bile acids with them. The useof such resins at best, however, only lowers serum cholesterol levels byabout 20%, and is associated with gastrointestinal side-effects,including constipation and certain vitamin deficiencies. Moreover, sincethe resins bind other drugs, other oral medications must be taken atleast one hour before or four to six hours subsequent to ingestion ofthe resin; thus, complicating heart patient's drug regimens.

Statins are cholesterol lowering agents that block cholesterol synthesisby inhibiting HMGCoA reductase, the key enzyme involved in thecholesterol biosynthetic pathway. Statins, e.g., lovastatin (Mevacor®),simvastatin (Zocor®), pravastatin (Pravachol®), fluvastatin (Lescol®)and atorvastatin (Lipitor®), are sometimes used in combination withbile-acid-binding resins. Statins significantly reduce serum cholesteroland LDL-serum levels, and slow progression of coronary atherosclerosis.However, serum HDL cholesterol levels are only moderately increased. Themechanism of the LDL lowering effect may involve both reduction of VLDLconcentration and induction of cellular expression of LDL-receptor,leading to reduced production and/or increased catabolism of LDLs. Sideeffects, including liver and kidney dysfunction are associated with theuse of these drugs (The Physicians Desk Reference, 56^(th) Ed., 2002,Medical Economics).

Niacin (nicotinic acid) is a water soluble vitamin B-complex used as adietary supplement and antihyperlipidemic agent. Niacin diminishesproduction of VLDL and is effective at lowering LDL. In some cases, itis used in combination with bile-acid binding resins. Niacin canincrease HDL when used at adequate doses, however, its usefulness islimited by serious side effects when used at such high doses. Niaspan®is a form of extended-release niacin that produces fewer side effectsthan pure niacin. Niacin/Lovastatin (Nicostatin®) is a formulationcontaining both niacin and lovastatin and combines the benefits of eachdrug.

Fibrates are a class of lipid-lowering drugs used to treat various formsof hyperlipidemia (i.e., elevated serum triglycerides) that may also beassociated with hypercholesterolemia. Fibrates appear to reduce the VLDLfraction and modestly increase HDL, however the effect of these drugs onserum cholesterol is variable. In the United States, fibrates such asclofibrate (Atromid-S®), fenofibrate (Tricor®) and bezafibrate(Bezalip®) have been approved for use as antilipidemic drugs, but havenot received approval as hypercholesterolemia agents. For example,clofibrate is an antilipidemic agent that acts (via an unknownmechanism) to lower serum triglycerides by reducing the VLDL fraction.Although serum cholesterol may be reduced in certain patientsubpopulations, the biochemical response to the drug is variable, and isnot always possible to predict which patients will obtain favorableresults. Atromid-S® has not been shown to be effective for prevention ofcoronary heart disease. The chemically and pharmacologically relateddrug, gemfibrozil (Lopid®) is a lipid regulating agent that moderatelydecreases serum triglycerides and VLDL cholesterol, and moderatelyincreases HDL cholesterol—the HDL₂ and HDL₃ subfractions as well as bothApoA-I and A-II (i.e., the AI/AMT-HDL fraction). However, the lipidresponse is heterogeneous, especially among different patientpopulations. Moreover, while prevention of coronary heart disease wasobserved in male patients between 40-55 without history or symptoms ofexisting coronary heart disease, it is not clear to what extent thesefindings can be extrapolated to other patient populations (e.g., women,older and younger males). Indeed, no efficacy was observed in patientswith established coronary heart disease. Serious side-effects areassociated with the use of fibrates including toxicity such asmalignancy (especially gastrointestinal cancer), gallbladder disease andan increased incidence in non-coronary mortality.

Oral estrogen replacement therapy may be considered for moderatehypercholesterolemia in post-menopausal women. However, increases in HDLmay be accompanied with an increase in triglycerides. Estrogen treatmentis, of course, limited to a specific patient population (postmenopausalwomen) and is associated with serious side effects including inductionof malignant neoplasms, gall bladder disease, thromboembolic disease,hepatic adenoma, elevated blood pressure, glucose intolerance, andhypercalcemia.

Other agents useful for the treatment of hyperlipidemia includeezetimibe (Zetia®; Merck), which blocks or inhibits cholesterolabsorption. However, inhibitors of ezetimibe have been shown to exhibitcertain toxicities.

HDL, as well as recombinant forms of ApoA-I complexed with phospholipidscan serve as sinks/scavengers for apolar or amphipathic molecules, e.g.,cholesterol and derivatives (oxysterols, oxidized sterols, plantsterols, etc.), cholesterol esters, phospholipids and derivatives(oxidized phospholipids), triglycerides, oxidation products, andlipopolysaccharides (LPS) (see, e.g., Casas et al., 1995, J. Surg. Res.Nov 59(5):544-52). HDL can also serve as also a scavenger for TNF-alphaand other lymphokines. HDL can also serve as a carrier for human serumparaoxonases, e.g., PON-1,-2,-3. Paraoxonase, an esterase associatedwith HDL, is important for protecting cell components against oxidation.Oxidation of LDL, which occurs during oxidative stress, appears directlylinked to development of atherosclerosis (Aviram, 2000, Free Radic. Res.33 Suppl:S85-97). Paraoxonase appears to play a role in susceptibilityto atherosclerosis and cardiovascular disease (Aviram, 1999, Mol. Med.Today 5(9):381-86). Human serum paraoxonase (PON-1) is bound tohigh-density lipoproteins (HDLs). Its activity is inversely related toatherosclerosis. PON-1 hydrolyzes organophosphates and may protectagainst atherosclerosis by inhibition of the oxidation of HDL andlow-density lipoprotein (LDL) (Aviram, 1999, Mol. Med. Today5(9):381-86). Experimental studies suggest that this protection isassociated with the ability of PON-1 to hydrolyze specific lipidperoxides in oxidized lipoproteins. Interventions that preserve orenhance PON-1 activity may help to delay the onset of atherosclerosisand coronary heart disease.

HDL further has a role as an antithrombotic agent and fibrinogenreducer, and as an agent in hemorrhagic shock (Cockerill et al., WO01/13939, published Mar. 1, 2001). HDL, and ApoA-I in particular, hasbeen show to facilitate an exchange of lipopolysaccharide produced bysepsis into lipid particles comprising ApoA-I, resulting in thefunctional neutralization of the lipopolysaccharide (Wright et al.,WO9534289, published Dec. 21, 1995; Wright et al., U.S. Pat. No.5,928,624 issued Jul. 27, 1999; Wright et al., U.S. Pat. No. 5,932,536,issued Aug. 3, 1999).

There are a variety of methods available for making lipoproteincomplexes in vitro. U.S. Pat. Nos. 6,287,590 and 6,455,088 disclose amethod entailing co-lyophilization of apolipoprotein and lipid solutionsin organic solvent (or solvent mixtures) and formation of chargedlipoprotein complexes during hydration of the lyophilized powder.Lipoprotein complexes can also be formed by a detergent dialysis method;e.g., a mixture of a lipid, a lipoprotein and a detergent such ascholate is dialyzed and reconstituted to form a complex (see, e.g.,Jonas et al., 1986, Methods Enzymol. 128:553-82). Example 1 of U.S.publication 2004/0067873 discloses a cholate dispersion method, in whicha lipid dispersion is combined with cholate under conditions for formingmicelles, and these in turn are incubated with an apoliprotein solutionto form complexes. Ultimately, the cholate, which is toxic, has to beremoved, e.g., by dialysis, ultrafiltration or adsorption absorptiononto an affinity bead or resin. U.S. Pat. No. 6,306,433 discloseslipoprotein complex formation by subjecting a fluid mixture of a proteinand lipid to high pressure homogenization. However, proteins that aresensitive to high shear forces can lose activity when exposed to highpressure homogenization.

Thus, currently available manufacturing methods result in wastage ofstarting materials, such as protein degradation, and/or requirepurification of the resulting product, such as removal of a toxic agent,and thus are inefficient and costly. Additionally, preparations oflipoprotein complexes can be heterogeneous, containing a mixture ofcomplexes varying in size and in composition. See, e.g., U.S. Pat. No.5,876,968. Accordingly, there is a need to develop new methods forproduction of lipoprotein complexes that are efficient and yield morehomogeneous complexes, preferably having a high degree of purity. Suchprocesses could allow more economical production on a large scale whilegenerating a more uniform pharmaceutically acceptable product with fewerrisks of side effects due to contaminants.

Moreover, the therapeutic use of ApoA-I, ApoA-I_(M), ApoA-I_(P) andother variants, as well as reconstituted HDL, is presently limited,however, by the large amount of apolipoprotein required for therapeuticadministration and by the cost of protein production, considering thelow overall yield of production and the occurrence of proteindegradation in cultures of recombinantly expressed proteins. (See, e.g.,Mallory et al., 1987, J. Biol. Chem. 262(9):4241-4247; Schmidt et al.,1997, Protein Expression & Purification 10:226-236). It has beensuggested by early clinical trials that the dose range is between 1.5-4g of protein per infusion for treatment of cardiovascular diseases. Thenumber of infusions required for a full treatment is unknown. (See,e.g., Eriksson et al., 1999, Circulation 100(6):594-98; Carlson, 1995,Nutr. Metab. Cardiovasc. Dis. 5:85-91; Nanjee et al., 2000,Arterioscler. Thromb. Vasc. Biol. 20(9):2148-55; Nanjee et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(4):979-89; Nanjee et al., 1996,Arterioscler. Thromb. Vasc. Biol. 16(9):1203-14).

Recombinant human ApoA-I has been expressed in heterologous hosts,however, the yield of mature protein has been insufficient forlarge-scale therapeutic applications, especially when coupled topurification methods that further reduce yields and result in impureproduct.

Weinberg et al., 1988, J. Lipid Research 29:819-824, describes theseparation of apolipoproteins A-I, A-II and A-IV and their isoformspurified from human plasma by reverse phase high pressure liquidchromatography.

WO 2009/025754 describes protein separation and purification ofalpha-1-antitrypsin and ApoA-I from human plasma.

Hunter et al., 2009, Biotechnol. Prog. 25(2):446-453, describeslarge-scale purification of the ApoA-I Milano variant that isrecombinantly expressed in E. coli.

Caparon et al., 2009, Biotechnol. And Bioeng. 105(2):239-249 describesthe expression and purification of ApoA-I Milano from an E. coli hostwhich was genetically engineered to delete two host cell proteins inorder to reduce the levels of these proteins in the purifiedapolipoprotein product.

U.S. Pat. No. 6,090,921 describes purification of ApoA-I orapolipoprotein E (ApoE) from a fraction of human plasma containingApoA-I and ApoE using anion-exchange chromatography.

Brewer et al., 1986, Meth. Enzymol. 128:223-246 describes the isolationand characterization of apolipoproteins from human blood usingchromatographic techniques.

Weisweiler et al., 1987, Clinica Chimica Acta 169:249-254 describesisolation of ApoA-I and ApoA-II from human HDL using fast-protein liquidchromatography.

deSilva et al., 1990, J. Biol. Chem. 265(24):14292-14297 describes thepurification of apolipoprotein J by immunoaffinity chromatography andreverse phase high performance liquid chromatography.

Lipoproteins and lipoprotein complexes are currently being developed forclinical use, with clinical studies using different lipoprotein-basedagents establishing the feasibility of lipoprotein therapy (Tardif,2010, Journal of Clinical Lipidology 4:399-404). One study evaluatedautologous delipidated HDL (Waksman et aL, 2010, J Am. Coll. Cardiol.55:2727-2735). Another study evaluated ETC-216, a complex of recombinantApoA-I_(M) and palmitoyl-oleoyl-PC (POPC) (Nissen et al., 2003, JAMA290:2292-2300). CSL-111 is a reconstituted human ApoA-I purified fromplasma complexed with soybean phosphatidylcholine (SBPC) (Tardif et al.,2007, JAMA 297:1675-1682). Current exploratory drugs have shown efficacyin reducing the atherosclerotic plaque but the effect was accompanied bysecondary effects such as increase in transaminases or formation ofApoA-I antibodies (Nanjee et al., 1999, Arterioscler. Vasc. Throm. Biol.19:979-89; Nissen et al., 2003, JAMA 290:2292-2300; Spieker et al.,2002, Circulation 105:1399-1402; Nieuwdorp et al., 2004, Diabetologia51:1081-4; Drew et al., 2009, Circulation 119, 2103-11; Shaw et al.,2008, Circ. Res. 103:1084-91; Tardiff et al., 2007, JAMA 297:1675-1682;Waksman, 2008, Circulation 118:S 371; Cho, U.S. Pat. No. 7,273,849 B2,issued Sep. 25, 2007). For example, the ERASE clinical trial (Tardiff etal., 2007, JAMA 297:1675-1682) utilized two doses of CSL-111: 40 mg/kgand 80 mg/kg of ApoA-I. The 80 mg/kg dose group had to be stopped due toliver toxicity (as shown by serious transaminase elevation). Even in the40 mg/kg dose group several patient experience transaminase elevation.

The need therefore exists for safer drugs that are more efficacious inlowering serum cholesterol, increasing HDL serum levels, preventingand/or treating dyslipidemia and/or diseases, conditions and/ordisorders associated with dyslipidemia. There is a need in the art forlipoprotein formulations that are not associated with liver toxicity,and preferably induce only minimal (or no) increase in triglycerides,LDL-triglycerides, or VLDL-triglycerides, as well as for robustproduction methods that can be used to reliably make these lipoproteinformulations on a commercial scale.

4. SUMMARY

The present disclosure provides lipoprotein complexes, comprising aprotein fraction (e.g., an apolipoprotein fraction) and a lipidfraction, and populations thereof that are especially suited to treatingand/or preventing dyslipidemia and diseases, disorders and/or conditionsassociated with dyslipidemia. It has been discovered that populations ofcomplexes which have greater purity and/or homogeneity, and/or compriseparticular ratios of lipids and proteins, as described herein, haveincreased ability to mobilize cholesterol combined with reduced risk ofside effects.

The lipoprotein complexes comprise a protein fraction (e.g., anapolipoprotein fraction) and a lipid fraction (e.g., a phospholipidfraction). The protein fraction includes one or more lipid-bindingproteins, such as apolipoproteins, peptides, or apolipoprotein peptideanalogs or mimetics capable of mobilizing cholesterol when present in alipoprotein complex. Non-limiting examples of such apolipoproteins andapolipoprotein peptides include preproapoliproteins, preproApoA I,proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-IV,proApoA-IV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE,preproApoA-I_(M), proApoA-I_(M), ApoA-I_(M), preproApoA-Ip, proApoA-Ip,ApoA-Ip, preproApoA-I_(Z), proApoA-I_(Z), and ApoA-I_(Z). Theapolipoprotein(s) can be in the form of monomers, dimers, or trimers, ormixtures thereof. In a specific embodiment, the apolipoprotein fractionconsists essentially of ApoA-I, most preferably of a single isoform.ApoA-I in lipoprotein complexes can have at least 90% or at least 95%sequence identity to a protein corresponding to amino acids 25 to 267 ofSEQ ID NO:1. Optionally, ApoA-I further comprises an aspartic acid atthe position corresponding to the full length ApoA-I amino acid 25 ofSEQ ID NO:1 (and position 1 of the mature protein). Preferably, at least75%, at least 80%, at least 85%, at least 90% or at least 95% of theApoA-I is correctly processed, mature protein (i.e., lacking the signaland propeptide sequences) and not oxidized, deamidated and/or truncated.

The present disclosure also provides mammalian host cells engineered toexpress ApoA-I, cell cultures comprising ApoA-I, and methods ofproducing mature, biologically active ApoA-I. It has been discoveredthat it is possible to engineer mammalian host cells to express largequantities of mature ApoA-I, that is substantially free of both immatureApoA-I (proApoA-I) and truncated forms of ApoA-I generated by proteasedegradation. These results are surprising. First, host cell machineryfor protein processing could be expected to be overwhelmed byoverexpression of a heterologous protein such as ApoA-I, leading to theproduction of the unprocessed, immature protein. Second, ApoA-I secretedinto the culture medium is subject to degradation by proteases and yet,only low levels of truncated ApoA-I are observed in the culture medium.The mammalian host cells, cell culture and the methods of producingApoA-I are particularly suited to the production of mature protein,useful in therapeutic applications, in commercially relevant quantities.

As provided herein, a mammalian host cell is engineered to express aprotein that preferably comprises (or consists of) an amino acidsequence having at least 95%, at least 96%, at least 97%, at least 98%,at least 99%, or 100% identity to positions 25 to 267 of SEQ ID NO:1.The protein preferably has an aspartic acid residue at the positioncorresponding to position 25 of SEQ ID NO:1. The mammalian host cell canoptionally further secrete such a protein. In some instances, theprotein expressed and/or secreted by the mammalian host cell can furthercomprise an 18-amino acid signal sequence (MKAAVLTLAVLFLTGSQA, SEQ IDNO:2) and/or a 6-amino acid propeptide sequence (RHFWQQ, SEQ ID NO:3).In some instances, the host cell is engineered to express a proteincomprising an amino acid sequence having at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1.

The host cell can be from any mammalian cell line, including but notlimited to Chinese hamster ovary (e.g., CHO-K1 or CHO-S), VERO, BHK, BHK570, HeLa, COS-1, COS-7, MDCK, 293, 3T3, PC12 and W138, or from anamyeloma cell or cell line (e.g., a murine myeloma cell or cell line).

The mammalian host cell can further contain multiple copies of a nucleicacid encoding an ApoA-I protein, e.g., a protein comprising orconsisting of positions 25 to 267 of SEQ ID NO:1. For example, themammalian host cell can contain at least about 5, 6, 7, 8, or morecopies, and up to about 10, 11, 12, 13, or 14 copies of the nucleicacid. The nucleic acid can further be operably linked to a promotercapable of expressing the protein at a high level in the mammalian hostcell, such as, for example, a simian cytomegalovirus promoter or morespecifically, immediate early simian cytomegalovirus promoter.

The mammalian host cells are preferably capable of producing at leastabout 0.5, 1, 2, or 3 g/L ApoA-I in culture and/or up to about 20 g/LApoA-I in culture, e.g., up to 4, 5, 6, 7, 8, 9, 10, 12, or 15 g/LApoA-I in culture. The culture can be of any scale, ranging from about150 mL to about 500 L, 1000 L, 2000 L, 5000 L, 10,000 L, 25,000 L, or50,000 L or more. In varying embodiments, the culture volume can rangefrom 10 L to 50 L, from 50 L to 100 L, from 100 L to 150 L, from 150 Lto 200 L, from 200 L to 300 L, from 300 L to 500 L, from 500 L to 1000L, from 1000 L to 1500 L, from 1500 L to 2000 L, from 2000 L to 3000 L,from 3000 L to 5000 L, from 5000 L to 7500 L, from 7500 L to 10,000 L,from 10,000 L to 20,000 L, from 20,000 L to 40,000 L, from 30,000 L to50,000 L. In some instances, the culture is a large scale culture, suchas 15 L, 20 L, 25 L, 30 L, 50 L, 100 L, 200 L, 300 L, 500 L, 1000 L,5000 L, 10,000 L, 15,000 L, 20,000 L, 25,000 L, up to 50,000 L or more.

The mammalian host cells of the present disclosure can be grown inculture. Thus, the present disclosure further provides a mammalian cellculture, comprising a plurality of mammalian host cells as describedabove or in Section 6.1.2 below. The cell culture can include one ormore of the following features: (a) the culture (which is optionally alarge scale batch culture of at least 10 liters, at least 20 liters, atleast 30 liters, at least 50 liters, at least 100 liters, 300 L, 500 L,1000 L, 5000 L, 10,000 L, 15,000 L, 20,000 L, 25,000 L, up to 50,000 Lor a continuous culture of at least 10 liters, at least 20 liters, atleast 30 liters, at least 50 liters, at least 100 liters, 300 L, 500 L,1000 L, 5000 L, or up to 10,000 L) comprises at least about 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0 g/L or more of mature ApoA-I proteincomprising or consisting of an amino sequence corresponding to aminoacids 25 to 267 of SEQ ID NO:1; (b) at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theprotein in the culture medium is an ApoA-I protein lacking a signalsequence; (c) at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% of the protein in the culturemedium is a mature ApoA-I protein lacking a signal sequence and apropeptide sequence; and (d) at least 75%, at least 80%, at least 85%,at least 90%, at least 95% of the mature ApoA-I is not truncated,oxidized, or deamidated.

The present disclosure also provides methods of producing mature,biologically active ApoA-I. Generally, the methods comprise culturingany of the mammalian host cells described above or in Section 6.1.2under conditions in which ApoA-I is expressed and secreted. The methodcan further include a step of recovering from the supernatant of acultured mammalian host cell, and optionally purifying, the mature,biologically active ApoA-I (such as by the methods disclosed in Sections6.1.3 and 6.1.4 below).

ApoA-I obtained or obtainable by the methods described above can furtherbe complexed with lipid to form a lipoprotein complex as describedherein, and/or incorporated into pharmaceutical compositions intherapeutically effective amounts. The pharmaceutical compositionspreferably are phosphate buffered solutions that also contain sucroseand/or mannitol as excipients.

It has further been discovered that, by purifying ApoA-I using a newcombination of chromatography and filtration steps, large quantities ofpure ApoA-I can be produced that contains levels of host cell proteins,host cell DNA, endotoxins and truncated forms of the protein that arelow enough to confer one or more attributes, including reduced risk ofside effects and low or no toxicity, rendering the protein particularlysuitable for therapeutic uses. Preferably, ApoA-I purified by themethods described herein is produced recombinantly in mammalian cellsand is secreted into the growth medium. Accordingly, the presentdisclosure relates to a method of purifying ApoA-I comprising the stepsof: (a) contacting an ApoA-I containing solution with an anion exchangematrix under conditions such that the ApoA-I does not bind to thematrix; (b) filtering the ApoA-I containing solution obtained in step(a) through a membrane having a pore size sufficient to remove virusesor viral particles; (c) passing the filtrate obtained in step (b)through a first reverse phase chromatography column under conditionssuch that the ApoA-I binds to the matrix; (d) eluting from the firstreverse phase chromatography matrix a first ApoA-I containing reversephase eluate using a gradient of increasing concentrations of an organicsolvent; (e) passing the first ApoA-I reverse phase eluate from step (d)through a second reverse phase chromatography column under conditionssuch that the ApoA-I binds to the matrix; and (f) eluting from thesecond reverse phase chromatography matrix a second ApoA-I containingreverse phase eluate using a gradient of increasing concentrations of anorganic solvent. The order in which the steps are performed is notcritical, for example, in an exemplary embodiment the step of filteringthrough a membrane to remove viruses or viral particles is performedafter step (f) above, rather than after step (a).

Also provided herein is a substantially pure ApoA-I product obtained orobtainable by the purification methods described herein in which theconcentration of ApoA-I is at least 10 g/L. The substantially pureApoA-I product produced by the purification methods described hereinpreferably comprises less than about 10 pg of host cell DNA per mg ofApoA-I, less than about 100 ng of host cell proteins per mg of ApoA-I,and/or less than 0.1 EU of endotoxin per mg of ApoA-I. The ApoA-Iproduct can be at least 95% pure, at least 96% pure, at least 97% pure,at least 98% pure or at least 99% pure.

Furthermore, the substantially pure ApoA-I product can be incorporatedinto any of the pharmaceutical compositions and/or lipoprotein complexesdescribed herein that comprise ApoA-I.

The lipid fraction typically includes one or more phospholipids whichcan be neutral, negatively charged, positively charged, or a combinationthereof. The fatty acid chains on phospholipids are preferably from 12to 26 or 16 to 26 carbons in length and can vary in degree of saturationfrom saturated to mono-unsaturated. Exemplary phospholipids includesmall alkyl chain phospholipids, egg phosphatidylcholine, soybeanphosphatidylcholine, dipalmitoylphosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerolssuch as dimyristoylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid,dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoylsphingomyelin, phytosphingomyelin, dipalmitoylsphingomyelin,distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives. Phospholipid fractionsincluding SM and palmitoylsphingomyelin can optionally include smallquantities of any type of lipid, including but not limited tolysophospholipids, sphingomyelins other than palmitoylsphingomyelin,galactocerebroside, gangliosides, cerebrosides, glycerides,triglycerides, and cholesterol and its derivatives.

Most preferably the lipid fraction contains at least one neutralphospholipid and, optionally, one or more negatively chargedphospholipids. In lipoprotein complexes that include both neutral andnegatively charged phospholipids, the neutral and negatively chargedphospholipids can have fatty acid chains with the same or differentnumber of carbons and the same or different degree of saturation. Insome instances, the neutral and negatively charged phospholipids willhave the same acyl tail, for example a C16:0, or palmitoyl, acyl chain.

When the lipid component of the complex comprises or consists of neutraland negatively charged phospholipids, the weight-to-weight (wt:wt) ratioof neutral to negatively charged phospholipid(s) is preferably in awt:wt ratio ranging from about 99:1 to about 90:10, more preferably fromabout 99:1 to about 95:5, and most preferably from about 98:2 to about96:4. In one embodiment, the neutral phospholipid(s) and the negativelycharged phospholipid(s) are present in a weight-to-weight (wt:wt) ratioof neutral phospholipid(s) to negatively charged phospholipid(s) ofabout 97:3.

The neutral phospholipid can be natural or synthetic. Preferably, thephospholipid is a sphingomyelin (“SM”), such as palmitoyl sphingomyelin,a phytosphingomyelin, a diphytosphingomyelin, a phosphosphingolipid, ora glycosphingolipid, optionally with saturated or mono-unsaturated fattyacids with chain lengths from 16 to 26 carbon atoms. SM can be from anysource. For example, the SM can be obtained from milk, egg, brain, ormade synthetically. In a specific embodiment, the SM is obtained fromchicken egg (“egg SM”). In another specific embodiment, the SM ispalmitoylsphingomyelin.

Any phospholipid that bears at least a partial negative charge atphysiological pH can be used as the negatively charged phospholipid.Non-limiting examples include negatively charged forms, e.g., salts, ofphosphatidylinositol, a phosphatidylserine, a phosphatidylglycerol and aphosphatidic acid. In a specific embodiment, the negatively chargedphospholipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],or DPPG, a phosphatidylglycerol. Preferred salts include potassium andsodium salts.

The phospholipids used to manufacture the complexes of the disclosureare preferably at least 95% pure and are more preferably at least 99%pure, and/or have levels of oxidation levels under 4 meq O/kg, morepreferably under 3 meq O/kg (e.g., under 2 meq O/kg). The major initialreaction product of oxygen and fatty acids is hydroperoxide. Using aniodometric method, it is possible to measure oxidation levels byassaying the presence of peroxide in a sample.

The lipoprotein complexes of the present disclosure preferably haveratios of apolipoprotein to lipid that result in more complete andhomogeneous complex formation, as shown in the examples below. Thelipoprotein complexes are characterized by an apolipoproteinfraction:lipid fraction molar ratio ranging from 1:80 to 1:120, from1:100 to 1:115, or from 1:105 to 1:110, where the apolipoprotein isexpressed in ApoA-I equivalents. In specific embodiments, the molarratio of the apolipoprotein fraction:lipid fraction is 1:80 to 1:90(e.g., 1:82, 1:85 or 1:87), from 1:90 to 1:100 (e.g., 1:95 or 1:98),from 1:100 to 1:110 (e.g., 1:105 or 1:108).

In specific embodiments, particularly those in which egg SM is used asthe neutral lipid, the weight ratio of the apolipoprotein fraction:lipidfraction ranges from about 1:2.7 to about 1:3 (e.g., 1:2.7).

The lipoprotein complexes of the present disclosure can also be used ascarriers to deliver hydrophobic, lipophilic or apolar active agents. Forsuch applications, the lipid fraction can further include, or thelipoprotein complex can be loaded with, one or more hydrophobic,lipophilic or apolar active agents, including but not limited to fattyacids, drugs, nucleic acids, vitamins, and/or nutrients. Specificexamples of active agents are described in Section 6.2.

The present disclosure also provides populations of the lipoproteincomplexes. Typically:

-   -   the populations contain a plurality of lipoprotein complexes,        each comprising a protein fraction and a lipid fraction;    -   the protein fractions contain a lipoprotein or lipoprotein        analog as described above, and in Section 6.1 or in Section        6.5.3; most preferably a protein fraction comprises or consists        essentially of lipoprotein (e.g., ApoA-I protein) that is        obtained or obtainable by the methods described in Section 6.1.2        and/or purified by the methods described in Section 6.1.4;    -   the lipid fractions contain a lipid as described above, and in        Section 6.2 or in Section 6.5.2;    -   the lipoprotein complexes are preferably produced by the thermal        cycling methods described in Section 6.5.4.

Applicants have discovered several features that are thought tocontribute individually or in combination to the potency and the safetyprofile of lipoprotein complex populations. These features include:

-   -   the homogeneity in the size of the complexes in a population,        mostly ranging between 4 nm and 15 nm (e.g., between 5 nm and 12        nm, between 6 nm and 15 nm, or between 8 nm and 10 nm);    -   the purity of the apolipoprotein used to make the complex (e.g.,        lack of oxidized, deamidated, truncated, and/or immature forms        of apolipoprotein and/or lack of endotoxin, and/or lack of        proteins other than apolipoprotein(s) (such as host cell        proteins), and/or host cell DNA that are often present in        recombinant production);    -   the purity of the complexes themselves in the population        (characterized by the lack of contaminants, such as solvents or        detergents use to prepare the complexes; the lack of oxidized        lipids; the lack of deamidated, oxidized or truncated proteins;        and/or reduced amounts or lack of uncomplexed apolipoprotein        and/or lipids).

Of these features, the homogeneity of the complexes and the prevalenceof mature, unmodified apolipoprotein in complexes, is thought toincrease potency. Purity of apolipoproteins, lipids, and the complexesreduces the risk of side effects such as liver damage reflected byincreases in liver enzymes (e.g., transaminases). Additionally, theinventors have made it feasible to make populations of lipoproteincomplexes by methods that result in the incorporation of most of theapolipoprotein into complexes, and the reduction in the amount ofuncomplexed apolipoprotein is also beneficial in that it reduces therisk of an immunogenic response in a subject that could be caused by theadministration of a heterologous protein.

Accordingly, the present disclosure provides populations of lipoproteincomplexes that are characterized by one or more, or even all, of thefollowing features:

-   -   (a) at least 75%, at least 80%, at least 85%, at least 90%, or        at least 95% by weight of the lipoprotein, typically ApoA-I, in        said population is in mature form;    -   (b) no more than 25%, no more than 20%, no more than 15%, no        more than 10% or no more than 5% by weight of the lipoprotein,        typically ApoA-I, in said population is in immature form;    -   (c) the population contains no more than 100 picograms, no more        than 50 picograms, no more than 25 picograms, no more than 10        picograms or no more than 5 picograms host cell DNA per        milligram of the lipoprotein, typically ApoA-I;    -   (d) the population contains no more than 500 nanograms, no more        than 200 nanograms, no more than 100 nanograms, no more than 50        nanograms, or no more than 20 nanograms host cell protein per        milligram of the lipoprotein, typically ApoA-I;    -   (e) no more than 25%, no more than 20%, no more than 15%, no        more than 10% or no more than 5% by weight of the lipoprotein,        typically ApoA-I, in the population is in truncated form;    -   (f) the lipoprotein component comprises or consists of mature        ApoA-I, and no more than 20%, no more than 15%, no more than        10%, no more than 5%, no more than 3%, no more than 2% or no        more than 1% of each of methionine 112 and methionine 148 in        said ApoA-I in said population is oxidized;    -   (g) at least 80%, at least 85%, at least 90% or at least 95% of        the lipoprotein complexes are in the form of particles of 4 nm        to 15 nm in size, e.g., 6 nm to 15 nm in size, or 8 to 12 nm in        size, yet more preferably 5 nm to 12 nm in size, as measured by        gel permeation chromatography (“GPC”) or dynamic light        scattering (“DLS”);    -   (i) the population contains no more than 1 EU, no more than 0.5        EU, no more than 0.3 EU or no more than 0.1 EU of endotoxin per        milligram of the lipoprotein, typically ApoA-I;    -   (j) no more than 15%, no more than 10%, no more than 5%, no more        than 4%, no more than 3%, no more than 2% or no more than 1% of        the amino acids in the lipoprotein, typically ApoA-I, in said        population is deamidated;    -   (k) no more than 15%, no more than 10%, no more than 5%, no more        than 2% or 0% by weight of the lipid in the lipid fraction in        said complexes is cholesterol;

(l) the population contains no more than 200 ppm, 100 ppm, 50 ppm of anon-aqueous solvent;

-   -   (m) the population does not contain any detergent (e.g.,        cholate);    -   (n) the population can be at least 80%, at least 85%, at least        90%, at least 95%, at least 97%, at least 98%, or at least 99%        homogeneous, as measured by percent of the population in a        single peak in gel permeation chromatography;    -   (o) the population is in a composition in which at least 80%, at        least 85%, at least 90%, at least 95% or at least 97% of the        protein is in complexed form;    -   (p) no more than 5%, no more than 4%, no more than 3%, no more        than 2% or no more than 1% of the lipid in said population is        oxidized; and    -   (q) no more than 15%, no more than 10%, no more than 5%, no more        than 4%, no more than 3%, no more than 2% or no more than 1% of        methionine and/or tryptophan residues in said population are        oxidized.

In specific embodiments, the population has features selected from thefollowing groups:

Group I: features (a), (b) and (e) above;

Group II: features (c), (d) and (i) above;

Group III: features (f), (j), (e), (p) and (q) above;

Group IV: features (g), (n) and (o) above;

Group V: features (l) and (m) above; and

Group VI: feature (k) above.

In certain aspects, the population is characterized by one or twofeatures independently selected from each of Group I and Group IV;optionally, the population is characterized by three featuresindependently selected from each of Group I and Group IV. The populationcan additionally be characterized by one, two or three featuresindependently selected from each of Group II and/or Group III. Thepopulation can yet be further characterized one or two featuresindependently selected from Group V and/or one feature independentlyselected from Group VI.

Certain lipid and protein components can form a plurality of differentbut homogeneous lipoprotein complexes. Accordingly, the presentdisclosure also provides compositions comprising two, three, or fourpopulations of lipoprotein complexes comprising different amounts ofapolipoprotein molecules (e.g., two, three or four ApoA-I molecules orApoA-I equivalents). In an exemplary embodiment, a composition comprisestwo lipoprotein complex populations, a first population comprisinglipoprotein complexes having 2 ApoA-I molecules or ApoA-I equivalentsper lipoprotein complex, a second population comprising lipoproteincomplexes having 3 or 4 ApoA-I molecules or ApoA-I equivalents perlipoprotein complex and optionally a third population comprisinglipoprotein complexes having 4 or 3 ApoA-I molecules or ApoA-Iequivalents per lipoproprotein complex, respectively.

The compositions comprising two or more populations of lipoproteincomplexes preferably have low levels of uncomplexed lipoprotein and/orlipid. Accordingly, preferably no more than 15%, no more than 12%, than10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%,no more than 5%, no more than 4%, no more than 3%, no more than 2%, orno more than 1% of the lipid in the composition is in uncomplexed formand/or no more than 15%, no more than 12%, no more than 10%, no morethan 9%, no more than 8%, no more than 7%, no more than 6%, no more than5%, no more than 4%, no more than 3%, no more than 2%, or no more than1% of the lipoprotein in the composition is in uncomplexed form.

The disclosure provides methods for making lipoprotein complexes. Themethods are based, inter alia, on the discovery that subjecting asuspension containing uncomplexed lipids and lipid-binding proteins orpeptides to thermal cycling conditions results in the formation oflipoprotein complexes with advantageous results relative to othermethods, such those in which lipoprotein complexes are produced byincubating the components at a fixed temperature.

The present disclosure provides thermal-cycling methods for preparinglipoprotein complexes, such as those described in Sections 6.5.1 to6.5.4. The methods typically comprise subjecting a suspension comprisinglipid particles (a “lipid component”) and lipid-binding proteins orpeptides (a “protein component”) to a plurality of thermal cycles untilmost of the protein component is incorporated into lipoproteincomplexes. The methods generally entail cycling the suspension between atemperature in a first, higher, temperature range and a temperature in asecond, lower, temperature range until lipoprotein complexes are formed.The high and low temperature ranges of the thermocycling process arebased on the phase transition temperatures of the lipid and proteincomponents of the lipoprotein complexes. Alternatively, where the lipidcomponent does not exhibit a defined or discrete phase transition, ascould occur when using phospholipids having unsaturated fatty acidchains or a mixture of phospholipids, the high and low temperatureranges of the thermocycling differ by at least about 20° C., up to about40° C. or even more. For example, in some embodiments, the low and hightemperature ranges differ by 20° C.-30° C., 20° C.-40° C., 20° C.-50°C., 30° C.-40° C., 30° C.-50° C., 25° C.-45° C., 35° C.-55° C.

For a lipid, the phase transition involves a change from a closelypacked, ordered structure, known as the gel state, to a loosely packed,less-ordered structure, known as the fluid state. Lipoprotein complexesare typically formed in the art by incubating lipid particles andapolipoproteins at temperatures near the transition temperature of theparticular lipid or mixture of lipids used. The phase transitiontemperature of the lipid component (which can be determined bycalorimetry) +/−12° C., more preferably +/−10° C., represents the “low”temperature range in the methods of the disclosure. In certainembodiments, the low temperature range is +/−3° C., +/−5° C., or +/−8°C. of the phase transition temperature of the lipid component. In onespecific embodiment, the low temperature range is from no less than 5°C. or no less than 10° C. below to 5° C. above the phase transitiontemperature of the lipid component.

For a protein, the phase transition temperature involves a change fromthe tertiary structure into the secondary structure. The phasetransition temperature of the protein component +/−12° C., morepreferably +/−10° C., represents the “high” temperature range in themethods of the disclosure. In specific embodiments, the high temperaturerange is +/−3° C., +/−5° C., or +/−8° C. of the phase transitiontemperature of the protein component. In one specific embodiment, thelow temperature range is from 10° C. below to no more than 5° C., nomore than 10° C., or no more than 15° C. above the phase transitiontemperature of the protein component.

The lipid component of the starting suspension, i.e., a suspension thathas not yet been subjected to thermal cycling, preferably comprisesparticles of lipids, e.g., is predominantly composed of lipids that arenot complexed to lipid-binding proteins. The make-up of the lipidcomponent is generally as described in Section 6.5.2 below.

The protein component of the starting suspension preferably containslipid-binding peptides and/or proteins that are uncomplexed to lipids,or are combined with lipids in protein/peptide to lipid ratio that is atleast 5-fold greater (e.g., at least 5-fold, at least 10-fold or atleast 20-fold greater) than the protein/peptide to lipid ratio in thedesired complex. The make-up of the protein component is generally asdescribed in Section 6.1 and in Section 6.5.3 below. The proteincomponent is preferably made according to the methods described inSection 6.1.2 and/or purified according to the methods described inSection 6.1.3 or 6.1.4.

In the methods of the disclosure, a suspension containing the proteincomponent and lipid component is typically thermally cycled between thehigh temperature range and the low temperature range, preferablystarting at a temperature in the high temperature range, untillipoprotein complexes are formed. Using suitable quantities of lipid andprotein components (e.g., as described in U.S. Patent Publication No.2006-0217312 A1, the contents of which are incorporated herein byreference in their entireties), substantially complete complexation ofthe lipid and protein components can be reached after several cycles.Further details of the protein and lipid stoichiometry suitable forthermal cycling methods are described in Section 6.5.4.

The complexes produced by the methods are typically supramolecularassemblies shaped as micelles, vesicles, spherical or discoidalparticles in which the protein component is physically bound to thelipid component at a specific stoichiometric range and with a homogenoussize distribution. The present methods advantageously result insubstantially complete complexation of the lipids and/or proteins in thestarting suspension, resulting in a composition that is substantiallywithout free lipids and/or free protein, as observed by separationmethods such as chromatography. Thus, the methods of the disclosure canbe performed in the absence of a purification step.

The lipid component in the starting suspension is typically in particleform. It is preferred that the particles are predominantly at least 45nm, at least 50 nm, at least 55 nm or at least 60 nm in size ranging upto 65 nm, up to 70 nm, up to 75 nm, up to 80 nm in size, up to 100 nm,up to 120 nm, up to 150 nm, up to 200 nm, up to 250 nm, up to 300 nm, upto 500 nm, for example, in the 45 nm to 100 nm or 45 to 250 nm sizerange, more preferably in the 50 nm to 90 nm size range, and mostpreferably in the 55 nm to 75 nm size range. In a preferred embodiment,the lipid particles are predominantly composed of egg-sphingomyelin andare 55 to 75 nm in size. In another preferred embodiment, the lipidparticles are predominantly composed of one or more syntheticsphingomyelin (e.g., palmitoylsphingomyelin or phytosphingomyelin) andare 175 nm to 250 nm in size. In yet another preferred embodiment, thelipid particles are predominantly composed of one or more syntheticlipids (e.g., palmitoyl sphingomyelin or phytosphingomyelin) and are 250nm to 1000 nm in size. In yet another preferred embodiment, the lipidparticles are predominantly composed of one or more synthetic lipids(e.g., palmitoyl sphingomyelin or phytosphingomyelin) and are 1000 nm to4000 nm in size. The sizes referred to herein are zeta (Z) average sizesas determined by dynamic light scattering. High pressure homogenization,for example microfluidization, advantageously produces lipid particlesof suitable sizes. Other methods for forming particles are disclosed inSection 6.5.2 below, and can be used as an alternative tohomogenization. If such methods produce particles outside the preferredsize ranges, the particles can be subject to size filtration to obtainparticles of a suitable size.

The methods of preparing lipoprotein complexes described hereinadvantageously produce complexes that are homogeneous in their sizedistribution, circumventing the need for size fractionation. Moreover,the methods of the disclosure result in substantially completeincorporation (e.g., at least 95%, at least 96%, at least 97%, at least98% or at least 99%) of the starting protein into lipoprotein particles.Accordingly, the disclosure provides a composition comprisinglipoprotein complexes and in which at least 95%, at least 96%, at least97%, at least 98% or at least 99% of the lipid-binding protein in thecomposition is complexed to lipid, for example as determined using gelpermeation chromatography. In specific embodiments, the disclosureprovides a composition comprising lipoprotein complexes which have 4 nmto 20 nm zeta average size, e.g., a 4 nm to 20 nm zeta average size, a 4nm to 15 nm zeta average size, a 4 nm to 12 nm zeta average size, a 5 nmto 15 nm zeta average size, a 5 nm to 12 nm zeta average size, a 5 nm to10 nm zeta average size, a 5 nm to 20 nm zeta average size, a 6 nm to 15nm zeta average size, or an 8 nm to 12 nm zeta average size, and inwhich at least 95%, at least 96%, at least 97%, at least 98% or at least99% of the lipid-binding protein in the composition is complexed tolipid, for example as determined using gel permeation chromatography.Subjecting lipid particles to thermal cycling with a lipoproteinaccording to the methods described herein typically results in apopulation of lipoprotein particles of 4 nm to 15 nm in size, forexample a population of lipoprotein particles of 6 nm to 15 nm in size,5 nm to 12 nm in size, or 8 nm to 12 nm in size. The size of the lipidparticles subjected to thermal cycling can range from 50 nm to 250 nm.In a preferred embodiment, the lipid particles are predominantlycomposed of egg-sphingomyelin and are 55 to 75 nm in size. In anotherpreferred embodiment, the lipid particles are predominantly composed ofone or more synthetic sphingomyelin (e.g., phytosphingomyelin) and are175 nm to 250 nm in size.

One or more steps in the methods of preparing lipoprotein complexes canbe carried out under an inert gas. Doing so can reduce or preventoxidation of apolipoproteins and/or lipids, thereby reducing risk ofside effects, such as liver damage. Suitable inert gases includenitrogen, helium, and argon.

The lipoprotein complexes obtained or obtainable by the methodsdescribed above are particularly suited to therapeutic uses because nofurther purification step is needed after the complexes are formed.

The present disclosure also provides populations of lipoproteincomplexes, and pharmaceutical compositions comprising lipoproteincomplexes or populations thereof, as described herein, and mayoptionally include one or more pharmaceutically acceptable carriers,excipients and/or diluents. In some embodiments, the pharmaceuticalcompositions are packaged in unit dosage amounts suitable foradministration. For example, in some embodiments, the compositionscomprise unit dosage amounts of dried (for example lyophilized)lipoprotein complexes packaged in sealed vials. Such compositions aresuitable for reconstitution with water, physiological solution (such assaline) or buffer, and administration via injection. Such compositionsmay optionally include one or more anti-caking and/or anti-agglomeratingagents to facilitate reconstitution of the charged complexes, or one ormore buffering agents, isotonicity agents (e.g., sucrose and/ormannitol), sugars or salts (e.g., sodium chloride) designed to adjustthe pH, osmolality and/or salinity of the reconstituted suspension. Thepopulations of lipoprotein complexes and/or pharmaceutical compositionsdescribed above can be manufactured under conditions that minimizeoxidation, thereby reducing the risk of side effects, such as liverdamage, caused by oxidized products. For example, pharmaceuticalcompositions can be manufactured under an inert gas, such as nitrogen,helium, or argon.

For commercial applications, it is useful to make large-scalepreparations of the lipoprotein complexes and pharmaceuticalcompositions. Accordingly, the present disclosure also provides apreparation of at least 1 L, 2 L, 5 L or 10 L and up to 15 L, 20 L, 30L, 50 L, or more (e.g., a preparation of 5 L to 30 L, 10 L to 15 L, or30 L to 50 L) comprising lipoprotein complexes in an amount sufficientto achieve a concentration of lipid-binding protein of at least about 3mg/mL, at least about 4 mg/mL, or at least about 5 mg/mL, and up toabout 10 mg/mL, about 15 mg/mL, or about 20 mg/mL, preferably rangingfrom about 8 mg/mL to about 12 mg/mL, most preferably about 8 mg/mL. Ina specific embodiment, the preparation has a volume of 15 L to 25 L andcontains about 100 g to about 250 g of ApoA-I. In another specificembodiment, the preparation has a volume of 30 L to 50 L and containsabout 240 g to about 780 g of ApoA-I.

The lipoprotein complexes described herein are useful to treatdyslipidemic disorders in animals, most preferably in humans. Suchconditions include, but are not limited to hyperlipidemia, andespecially hypercholesterolemia (including heterozygous and homozygousfamilial hypercholesterolemia), and cardiovascular disease such asatherosclerosis (including treatment and prevention of atherosclerosis)and the myriad clinical manifestations of atherosclerosis, such as, forexample, stroke, ischemic stroke, transient ischemic attack, myocardialinfarction, acute coronary syndrome, angina pectoris, intermittentclaudication, critical limb ischemia, valve stenosis, and atrial valvesclerosis; restenosis (e.g., preventing or treating atheroscleroticplaques which develop as a consequence of medical procedures such asballoon angioplasty); and other disorders, such as endotoxemia, whichoften results in septic shock.

Lipoprotein complexes and compositions as described herein have beenfound to effect and/or facilitate cholesterol efflux when administeredat doses lower than those used for other lipoprotein complexes instudies to date. See, e.g., Spieker et al., 2002, Circulation105:1399-1402 (using a dose of 80 mg/kg); Nissen et al., 2003, JAMA290:2292-2300 (using doses of 15 mg/kg or 40 mg/kg); Tardif et al., 2007JAMA 297:1675-1682 (using doses of 40 mg/kg to 80 mg/kg), using dosesthat range from 15 mg/kg to 80 mg/kg. Additionally, the lipoproteincomplexes as described herein have been found to have reduced sideeffects. As shown in the examples below, it has been discovered thatlipoprotein complexes of the disclosure effectively mobilize cholesterolat doses as low as 2 mg/kg, and, in contrast lipoprotein complexespreviously administered to human patients, do not significantly raisethe levels of triglycerides, VLDL, and liver enzymes such astransaminases Nanjee et al., 1999, Arterioscler. Vasc. Throm. Biol.19:979-89). Moreover, the reduced side effects are observed even asdoses are increased up to about 15 mg/kg (lack of triglycerideelevation) or even as high as 45 mg/kg (lack of transaminase increase)in normal subjects with normal liver and/or kidney function. Thus, theability of the complexes of the present disclosure to be administeredwithout side effects is preferably assessed in an individual with normalliver function, normal kidney function, or both.

Without being bound by theory, the inventors attribute the benefits ofthe lipoprotein complexes of the disclosure to a more homogeneous sizedistribution of complexes and reduced amounts of damaged protein and/orlipid (e.g., oxidized protein, deamidated protein, and oxidized lipid)as compared to prior treatments. It is further believed that thenegatively charged phospholipids comprising the lipid fraction willimpart the complexes and compositions described herein with improvedtherapeutic properties over conventional lipoprotein complexes. One ofthe key differences between small discoidal pre-beta HDL particles,which are degraded in the kidney, and large discoidal and/or sphericalHDL, which are recognized by the liver where their cholesterol is eitherstored, recycled, metabolized (as bile acids) or eliminated (in thebile), is the charge of the particles. The small, discoidal pre-beta HDLparticles have a lower negative surface charge than large, discoidaland/or spherical HDL particles that are negatively charged. It isbelieved that the higher negative charge is one of the factors thattrigger the recognition of the particles by the liver, and thattherefore avoids catabolism of the particles by the kidney. Furthermore,it has been shown that the kidney does not absorb readily absorb chargedparticles (see Hacker et al., 2009, Pharmacology: Principles andPractice, 183). Thus, owing in part to the presence of the chargedphospholipids(s), it is believed that negatively charged lipoproteincomplexes and compositions described herein will stay in the circulationlonger than conventional lipoprotein complexes, or that the charge willaffect the half-life of the lipoprotein in a charge-dependent manner. Itis expected that their longer circulation (residence) time, combinedwith a reduction in the rate and/or extent to which the complexesaggregate and fuse with existing HDL as a result of the negative charge,will facilitate cholesterol mobilization (by giving the complexes moretime to accumulate cholesterol) and esterification (by providing moretime for the LCAT to catalyze the esterification reaction). The chargemay also increase the rate of cholesterol capture and/or removal,thereby facilitating removal of cholesterol in larger quantities. As aconsequence, it is expected that the negatively charged lipoproteincomplexes and compositions described herein will provide therapeuticbenefit over conventional lipoprotein therapies, as less complex and/orcomposition will need to be administered, and less often, with reducedside effects.

Accordingly, the methods provided herein generally involve administeringto a subject a therapeutically effective amount of a lipoproteincomplex, a population of lipoprotein complexes, or pharmaceuticalcomposition described herein to treat or prevent a dyslipidemicdisorder. The lipoprotein complex can be administered at a dose rangingfrom about 0.25 mg/kg ApoA-I equivalents to about 45 mg/kg, e.g., a doseof about 0.5 mg/kg to about 30 mg/kg or about 1 mg/kg ApoA-I equivalentsup to about 15 mg/kg ApoA-I equivalents per injection. The dose canfurther be tailored to the individual being treated by selecting a dosethat minimizes the increase in the level of triglycerides,VLDL-cholesterol and/or VLDL-triglyceride. In specific embodiments, thedose is about 3 mg/kg, about 6 mg/kg, or about 12 mg/kg.

The methods further comprise administering the lipoprotein complex at aninterval ranging from 5 to 14 days, or from 6 to 12 days, such as aninterval of one or two weeks. The methods can further compriseadministering the lipoprotein complex 4, 5, 6, 7, 8, 9, 10, 11, 12, orup to 52 times at any of the intervals described above, and preferablyat an interval of one week. For example, in one embodiment, thelipoprotein complex is administered six times, with an interval of 1week between each administration. For chronic conditions, more than 52administrations can be carried out. Optionally, the methods can bepreceeded by an initial induction phase where the lipoprotein complex isadministered more frequently.

Complexes and/or pharmaceutical compositions thereof, can beadministered parenterally, e.g., intravenously. Intravenousadministration can be done as an infusion over a period of time rangingfrom about 1 to about 24 hours, or about 1 to 4 hours, about 0.5 to 2hours, or about 1 hour.

The examples below show small increases in triglyceride levels followingadministration of doses 30 mg/kg and 45 mg/kg, which is explained by theincrease in VLDL and LDL resulting from the high degree of cholesterolmobilization. Those parameters can be controlled during the treatment,as they are routinely measured in hospital laboratories with standardlipid panels. Based on examples below, the dose selection can beachieved to minimize the increase in the level of triglycerides andVLDL-cholesterol and VLDL-triglyceride dependent on the patient reactionto the medicine, which allows a personalized-type medicine.

The complexes and/or compositions can be administered alone (asmonotherapy) or, alternatively, they can be adjunctively administeredwith other therapeutic agents useful for treating and/or preventingdyslipidemia and/or its associated conditions, diseases and/ordisorders. Non-limiting examples of therapeutic agents with which thenegatively charged lipoprotein complexes and compositions describedherein can be adjunctively administered include bile acid-bindingresins, HMG CoA-reductase inhibitors (statins), niacin, resins,inhibitors of cholesterol absorption, platelet aggregation inhibitors,fibrates, anticoagulants, CETP inhibitors (e.g., anacetrapib anddalcetrapib), and/or PCSKG antibodies or ligands.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: the amino acid sequence of human prepro-Apolipoprotein A-I (SEQID NO: 1; GenBank Accession no. AAB59514.1). Amino acids 1-18, in boldfont, correspond to the signal sequence of ApoA-I, and amino acids 19-24correspond to the propeptide sequence, underlined. Both the signalsequence and the propeptide are cleaved in cells to produce thefull-length mature human ApoA-I (amino acids 25-267).

FIG. 2: ApoA-I titers in 12-day fed batch cultures of recombinant S-CHOcells expressing ApoA-I. Cells were continuously cultured by serialpassage from generation 0 to generation 43. The ApoA-I production wasmonitored in generations 4, 8, 14, 19, 25, 21, 36 and 43 by reversephase HPLC. The amount of ApoA-I in the culture medium varied from 1259mg/L to 1400 mg/L.

FIGS. 3A-3B: FIG. 3A shows the viable cell density in a 200 L 13-dayculture of recombinant S-CHO cells expressing ApoA-I. Viable celldensity peaked on day 9 at 33.20×105 cells/mL with a viability of 92.5%.FIG. 3B shows the concentration of ApoA-I in the culture medium of the13-day culture. The concentration of ApoA-I in the culture medium peakedon day 12 at around 2 mg/mL.

FIG. 4: SDS polyacrylamide gel of ApoA-I purified by the methodsdescribed herein. The left-hand lane shows molecular weight markers. Theright-hand lane shows purified ApoA-I having a molecular weight ofapproximately 28kD.

FIGS. 5A-5D: HPLC chromatograms of proApoA-I/SM complexes atprotein:lipid weight ratios of 1:2.5 (Formula A; FIG. 5A), 1:2.7(Formula B; FIG. 5B), 1:3.1 (Formula C; FIG. 5C), and proApoA-I/SM/DPPCcomplexes at a protein:lipid weight ratio of 1:2.7 (Formula D; FIG. 5D).

FIGS. 6A-6D: HPLC chromatograms of lipoprotein complexes of Formula D at10, 20, 30, and 60 minutes, respectively.

FIGS. 7A-7D: HPLC chromatograms of lipoprotein complexes of Formula B at10, 20, 30, and 50 minutes, respectively.

FIGS. 8A-8D: HPLC chromatograms of lipoprotein complexes of Formula F at20, 40, 60, and 120 minutes, respectively.

FIG. 9: Chart of pre-beta HDL complex formation over time illustratingformation of lipoprotein complexes with a 1:2.7 lipoprotein:totalphospholipid wt:wt ratio comprising proApoA-I and SM (Formula B),proApoA-I, SM, DPPC and DPPG with a SM:DPPC:DPPG wt:wt ratio of 48:48:4(Formula F), and proApoA-I, SM, DPPC and DPPG with a SM:DPPC:DPPG wt:wtratio of 73:23:4 (Formula G).

FIG. 10A-10D: HPLC chromatograms for Formulae E, H, I, and J,respectively.

FIG. 11: Schematic diagram of exemplary process for making lipoproteincomplexes.

FIG. 12: Exemplary thermal cycling apparatus used for non-commercialscale thermal cycling runs.

FIGS. 13A-13E: Gel permeation chromatogram of SM/DPPG/ApoA-I proteincomplexes with increasing number of thermal cycles. The components weresubject to thermal cycling between 57° C. and 37° C. for 5 minutes ateach temperature, for a total of 30 minutes (FIG. 13A), 60 minutes (FIG.13B), 120 minutes (FIG. 13C), 180 minutes (FIG. 13D) or 210 minutes(FIG. 13E).

FIG. 14: Gel permeation chromatogram of SM/ApoA-I complexes.

FIG. 15: Gel permeation chromatogram ofN-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of plant SM orphytosphingomyelin)/DPPG/ApoA-I complexes.

FIG. 16: Gel permeation chromatogram of synthetic palmitoylSM/DPPG/ApoA-I complexes.

FIG. 17: Gel permeation chromatogram of phytosphingomyelin/DPPG/ApoA-Icomplexes.

FIG. 18: Gel permeation chromatogram of SM/DPPC/DPPG/ApoA-I peptidecomplexes.

FIGS. 19A-19D: Characterization of lipid particles by a Dynamic LightScattering system using a Malvern Instruments Zetasizer (MalvernInstruments Inc.). In FIG. 19A, the Z average is 84.49 nm (the “84 nmparticles”); in FIG. 19B, the Z average is 76.76 nm (the “77 nmparticles”); in FIG. 19C, the Z average is 66.21 nm (the “66 nmparticles”); and in FIG. 19D, the Z average is 59.50 nm (the “60 nmparticles”).

FIGS. 20A-20D: Gel permeation chromatograms of complexes after fivethermal cycles with 84 nm (FIG. 20A), 77 nm (FIG. 20B), 66 nm (FIG. 20C)and 60 nm (FIG. 20D) lipid particles.

FIGS. 21A-21B: Gel permeation chromatograms of complexes after sixthermal cycles starting with 450 nm (FIG. 21A) and 40 nm (FIG. 21B)lipid particles.

FIG. 22: Gel permeation chromatogram of complexes after six thermalcycles starting with 65 nm lipid particles, with the first cycleinitiated at the “low temperature” of 37° C.

FIG. 23: Schematic diagram of exemplary embodiment for makingpharmaceutical compositions comprising lipoprotein complexes, whichincludes formulating the lipoprotein complexes produced by the methodsof the disclosure into commercially useful pharmaceutical compositions.

FIG. 24: Increase in plasma VLDL-total cholesterol levels followinginfusion of a lipoprotein complex according to Formula B and Formula H.Lipoprotein complexes according to Formula H (▪) and Formula B (▾) wereinfused into fasted rabbits at doses of 5 mg/kg. Baseline values,ranging from 0.03 to 0.3 g/L for the three groups, were subtracted todetermine the increase in plasma VLDL-total cholesterol levels.

FIG. 25: Increase in plasma triglyceride levels following infusion of alipoprotein complex according to Formula B and Formula H. Lipoproteincomplexes according to Formula H (▪) and Formula B (▴) were infused intofasted rabbits at doses of 5 mg/kg. Baseline values, ranging from 0.31to 0.71 g/L for the three groups, were subtracted to determine theincrease in plasma triglyceride levels.

FIG. 26A-26D: Increase in plasma total cholesterol (FIG. 26A),triglycerides (FIG. 26B), phospholipids (FIG. 26C), and ApoA-I (FIG.26D) following infusion of 5 mg/kg or 20 mg/kg of and 20 mg/kg inrabbits of ApoA-I/eggSM complexes (, ▴) or ApoA-I/synthetic SMcomplexes (♦, ▾), as compared to diluent (▪). Baseline values ranged asfollows for the different plasma lipids measured: from 0.28 to 0.4 g/Lfor plasma cholesterol, from 0.23 to 0.29 g/L for plasma triglycerides,and from 0.45 to 0.61 g/L for plasma phospholipids.

FIG. 27A-27C: Increase in plasma HDL-total cholesterol (FIG. 27A),LDL-total cholesterol (FIG. 27B), and VLDL-total cholesterol (FIG. 27C)following infusion in rabbits of 5 mg/kg ApoA-I/eggSM complexes () andApoA-I/synthetic SM complexes (♦) as compared to diluent (▪). Baselinevalues ranged as follows: between 0.20 to 0.31 g/L for plasma HDL-totalcholesterol, between 0.06 to 0.09 g/L for plasma LDL-total cholesterol,and between 0.007 to 0.011 g/L for plasma VLDL-total cholesterol.

6. DETAILED DESCRIPTION

The present disclosure provides lipoprotein complexes, populationsthereof, along with methods of making the lipoprotein complexes. Thecomplexes, and populations and compositions (e.g., pharmaceuticalcompositions) thereof, are useful for, among other things, the treatmentand/or prophylaxis of dyslipidemia and/or diseases, disorders and/orconditions associated with dyslipidemia. As discussed in the Summarysection, the lipoprotein complexes comprise two major fractions, anapolipoprotein fraction and a phospholipid fraction, preferably indefined weight or molar ratios, and preferably including a specifiedamount of a neutral phospholipid and, optionally, one or more negativelycharged phospholipids.

6.1. The Protein Fraction

The present disclosure provides lipoprotein complexes which comprise aprotein fraction. The present disclosure further provides methods ofmaking lipoprotein complexes. The protein component of the lipoproteincomplexes is not critical for success in the present methods. Virtuallyany lipid-binding protein, such as an apolipoprotein and/or derivativeor analog thereof that provides therapeutic and/or prophylactic benefitcan be included in the complexes. Moreover, any alpha-helical peptide orpeptide analog, or any other type of molecule that “mimics” the activityof an apolipoprotein (such as, for example ApoA-I) in that it canactivate LCAT or form discoidal particles when associated with lipids,can be included in the lipoprotein complexes, and is encompassed by theterm “lipid-binding protein.”

6.1.1. Lipid Binding Proteins

The present disclosure further provides methods of purifyingrecombinantly produced protein, e.g., for use in making lipoproteincomplexes. The recombinantly produced protein is most suitably anapolipoprotein. Suitable proteins include apolipoproteins ApoA-I,ApoA-II, ApoA-IV, ApoA-V and ApoE; preferably in mature form.Lipid-binding proteins also active polymorphic forms, isoforms, variantsand mutants as well as truncated forms of the foregoing apolipoproteins,the most common of which are Apolipoprotein A-I_(Milano) (ApoA-I_(M)),Apolipoprotein A-I_(Paris) (ApoA-I_(P)), and ApolipoproteinA-I_(Zaragoza) (ApoA-I_(Z)). Apolipoproteins mutants containing cysteineresidues are also known, and can also be used (see, e.g., U.S.Publication No. 2003/0181372). The apolipoproteins may be in the form ofmonomers or dimers, which may be homodimers or heterodimers. Forexample, homo- and heterodimers (where feasible) of ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-I_(M)(Franceschini et al., 1985, J. Biol. Chem. 260:1632-35), ApoA-I_(P)(Daum et al., 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al.,1985, J. Biol. Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol.Chem. 259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J.Biochem. 201(2):373-83), ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000), ApoJ and ApoH may be used. The apolipoproteins mayinclude residues corresponding to elements that facilitate theirisolation, such as His tags, or other elements designed for otherpurposes, so long as the apolipoprotein retains some biological activitywhen included in a complex.

Such apolipoproteins can be purified from animal sources (and inparticular from human sources) or produced recombinantly as iswell-known in the art, see, e.g., Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29. See alsoU.S. Pat. Nos. 5,059,528, 5,128,318, 6,617,134; U.S. Publication Nos.20002/0156007, 2004/0067873, 2004/0077541, and 2004/0266660; and PCTPublications Nos. WO/2008/104890 and WO/2007/023476. Other methods ofpurification are also possible, for example as described in Sections6.1.3 and 6.1.4 below.

Non-limiting examples of peptides and peptide analogs that correspond toapolipoproteins, as well as agonists that mimic the activity of ApoA-I,ApoA-I_(M), ApoA-II, ApoA-IV, and ApoE, that are suitable for use asapolipoproteins in the complexes and compositions described herein aredisclosed in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166 (issuedto Dasseux et al.), U.S. Pat. No. 5,840,688 (issued to Tso), U.S.Publication Nos. 2004/0266671, 2004/0254120, 2003/0171277 and2003/0045460 (to Fogelman), U.S. Publication No. 2003/0087819 (toBielicki) and PCT Publication No. WO/2010/093918 (to Dasseux et al.),the disclosures of which are incorporated herein by reference in theirentireties. These peptides and peptide analogues can be composed ofL-amino acid or D-amino acids or mixture of L- and D-amino acids. Theymay also include one or more non-peptide or amide linkages, such as oneor more well-known peptide/amide isosteres. Such “peptide and/or peptidemimetic” apolipoproteins can be synthesized or manufactured using anytechnique for peptide synthesis known in the art, including, e.g., thetechniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and6,046,166.

The complexes can include a single type of lipid-binding protein, ormixtures of two or more different lipid-binding proteins, which may bederived from the same or different species. Although not required, thelipoprotein complexes will preferably comprise lipid-binding proteinsthat are derived from, or correspond in amino acid sequence to, theanimal species being treated, in order to avoid inducing an immuneresponse to the therapy. Thus, for treatment of human patients,lipid-binding proteins of human origin are preferably used in thecomplexes of the disclosure. The use of peptide mimetic apolipoproteinsmay also reduce or avoid an immune response.

In certain preferred embodiments, the lipid-binding protein is a proteinhaving an amino acid sequence with at least 95% sequence identity to amature human ApoA-I protein, e.g., a protein having an amino acidsequence corresponding to positions 25 to 267 of SEQ ID NO:1. In certainembodiments, the mature human ApoA-I protein has an amino acid sequencewith at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to positions 25 to 267 of SEQ ID NO:1. In some embodiments, themature human ApoA-I protein has an amino acid sequence having anaspartic acid at position 1 (i.e. the position corresponding to position25 of SEQ ID NO:1). In a specific embodiment, the mature human ApoA-Iprotein has an amino acid sequence corresponding to positions 25 to 267of SEQ ID NO:1. In a preferred embodiment, the ApoA-I protein isrecombinantly produced in mammalian host cells, most preferably ChineseHamster Ovary (“CHO”) cells, as described in the following subsection.

6.1.2. Recombinant Expression of Apolipoproteins

The present disclosure provides recombinant expression methods forproducing lipid binding proteins such as ApoA-I, and related nucleicacids, mammalian host cells, cell cultures. The resulting recombinantlipid binding protein can be purified and/or incorporated intolipoprotein complexes as described herein.

Generally, for recombinant production, a polynucleotide sequenceencoding a lipid-binding protein or peptide is inserted into anappropriate expression vehicle, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedcoding sequence, or in the case of an RNA viral vector, the necessaryelements for replication and translation. The expression vector can bederived from viruses such as adenovirus, adeno-associated virus,herpesvirus, retrovirus or lentivirus. The expression vehicle is thentransfected into a suitable target cell which will express the proteinor peptide. Suitable host cells include, but are not limited to,bacterial species, mammalian or insect host cell systems includingbaculovirus systems (see, e.g., Luckow et al., Bio/Technology, 6, 47(1988)), and established cell lines such 293, COS-7, C127, 3T3, CHO,HeLa, BHK, etc. Depending on the expression system used, the expressedpeptide is then isolated by procedures well-established in the art.Methods for recombinant protein and peptide production are well known inthe art (see, e.g., Sambrook et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. each of which is incorporated byreference herein in its entirety.)

Where ApoA-I is the lipid binding protein, ApoA-I protein is expressedfrom a recombinant nucleotide sequence encoding ApoA-I. In someembodiments, the nucleotide sequence encoding ApoA-I is human.Non-limiting examples of human ApoA-I nucleotide sequences are disclosedin U.S. Pat. Nos. 5,876,968; 5,643,757; and 5,990,081, and WO 96/37608;the disclosures of which are incorporated herein by reference in theirentireties. In certain embodiments, the nucleotide sequence encodes theamino acid sequence of a mature ApoA-I protein, preferably operablylinked to a signal sequence (e.g., amino acids 1-18 of SEQ ID NO:1) forsecretion of the ApoA-I from the host cell and/or a proprotein sequence(e.g., amino acids 19-25 of SEQ ID NO:1). Other signal sequencessuitable for directed secretion of ApoA-I can be either heterologous toApoA-I, e.g., a human albumin signal peptide or a human IL-2 signalpeptide, or homologous to ApoA-I.

Preferably, the nucleotide sequence encodes a mature human ApoA-Ipolypeptide, for example a polypeptide having an amino acid sequencethat is at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical to the amino acid sequence corresponding topositions 25 to 267 of SEQ ID NO:1, optionally wherein the amino acidsequence comprises an aspartic acid at position 25. In a preferredembodiment, the nucleotide sequence encodes a polypeptide having anamino acid sequence of SEQ ID NO:1. The nucleotide sequence can alsoencode a polypeptide having an amino acid sequence that is at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identical tothe amino acid sequence of human ApoA-I protein set forth in one ofGenBank Accession Nos. NP 000030, AAB59514, P02647, CAA30377, AAA51746or AAH05380.1, optionally comprising an aspartic acid at the positioncorresponding to the first amino acid of the mature human ApoA-Iprotein.

The ApoA-I encoding polynucleotides can be codon optimized forexpression in recombinant host cells. Preferred host cells are mammalianhost cells, including, but not limited, Chinese hamster ovary cells(e.g. CHO-K1; ATCC No. CCL 61; CHO-S (GIBCO Life Technologies Inc.,Rockville, Md., Catalog #11619012)), VERO cells, BHK (ATCC No. CRL1632), BHK 570 (ATCC No. CRL 10314), HeLa cells, COS-1 (ATCC No. CRL1650), COS-7 (ATCC No. CRL 1651), MDCK cells, 293 cells (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), 3T3 cells, myelomacells (especially murine), PC12 cells and W138 cells. In certainembodiments, the mammalian cells, such as CHO-S cells (Invitrogen™,Carlsbad Calif.), are adapted for growth in serum-free medium.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va.

For recombinant expression of ApoA-I, the polynucleotides encodingApoA-I are operably linked to one or more control sequences, e.g., apromoter or terminator, that regulate the expression of ApoA-I in thehost cell of interest. The control sequence(s) can be native or foreignto the ApoA-I-encoding sequence, and also native or foreign to the hostcell in which the ApoA-I is expressed. Control sequences include, butare not limited to, promoters, ribosome binding sites, leaders,polyadenylation sequences, propeptide sequences, signal peptidesequences, and transcription terminators. In some embodiments, thecontrol sequences include a promoter, ribosome binding site, andtranscriptional and translational stop signals. The control sequencescan also include one or more linkers for the purpose of introducingspecific restriction sites facilitating ligation of the controlsequences with the coding region of the nucleotide sequence encodingApoA-I.

The promoters driving the recombinant expression of ApoA-I can beconstitutive promoters, regulated promoters, or inducible promoters.Appropriate promoter sequences can be obtained from genes encodingextracellular or intracellular polypeptides which are either endogenousor heterologous to the host cell. Methods for the isolation,identification and manipulation of promoters of varying strengths areavailable in or readily adapted from the art. See e.g., Nevoigt et al.(2006) Appl. Environ. Microbiol. 72:5266-5273, the disclosure of whichis herein incorporated by reference in its entirety.

One or more of the control sequences can be derived from viral sources.For example, in certain aspects, promoters are derived from polyoma oradenovirus major late promoter. In other aspects, the promoter isderived from Simian Virus 40 (SV40), which can be obtained as a fragmentthat also contains the SV40 viral origin of replication (Fiers et al.,1978, Nature, 273:113-120), or from cytomegalovirus, e.g., simiancytomegalovirus immediate early promoter. (See U.S. Pat. No. 4,956,288).Other suitable promoters include those from metallothionein genes (SeeU.S. Pat. Nos. 4,579,821 and 4,601,978).

Also provided herein are recombinant ApoA-I expression vectors. Arecombinant expression vector can be any vector, e.g., a plasmid or avirus, that can be manipulated by recombinant DNA techniques tofacilitate expression of a heterologous ApoA-I in a recombinant hostcell. The expression vector can be integrated into the chromosome of therecombinant host cell and comprises one or more heterologous genesoperably linked to one or more control sequences useful for productionof ApoA-I. In other embodiments, the expression vector is anextrachromosomal replicative DNA molecule, e.g., a linear or closedcircular plasmid, that is found either in low copy number (e.g., fromabout 1 to about 10 copies per genome equivalent) or in high copy number(e.g., more than about 10 copies per genome equivalent). In variousembodiments, the expression vector includes a selectable marker, such asa gene that confers antibiotic resistance (e.g., ampicillin, kanamycin,chloramphenicol or tetracycline resistance) to the recombinant hostorganism that comprises the vector. In particular aspects, the DNAconstructs, vectors and polynucleotides are suitable for expression ofApoA-I in mammalian cells. Vectors for expression of ApoA-I in mammaliancells can include an origin of replication, a promoter and any necessaryribosome binding sites, RNA splice sites, polyadenylation site, andtranscriptional terminator sequences that are compatible with the hostcell systems. In some aspects, an origin of replication is heterologousto the host cell, e.g., is of viral origin (e.g., SV40, Polyoma, Adeno,VSV, BPV). In other aspects, an origin of replication is provided by thehost cell chromosomal replication mechanism.

Methods, reagents and tools for introducing foreign DNA into mammalianhost cells are known in the art and include, but are not limited to,calcium phosphate-mediated transfection (Wigler et al., 1978, Cell14:725; Corsaro et al., 1981, Somatic Cell Genetics 7:603; Graham etal., 1973, Virology 52:456), electroporation (Neumann et al., 1982, EMBOJ. 1:841-5), DEAE-dextran mediated transfection (Ausubel et al. (eds.),Short Protocols in Molecular Biology, 3rd Edition (John Wiley & Sons1995)), and liposome-mediated transfection (Hawley-Nelson et al., 1993,Focus 15:73; Ciccarone et al., 1993, Focus 15:80).

For high-yield production, stable expression of ApoA-I is preferred. Forexample, following the introduction of foreign DNA into the host cells,the host cells may be allowed to grow for 1-2 days in an enriched media,and then are switched to a selective media. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with vector comprising a nucleotide sequence comprising theApoA-I-coding sequence controlled by appropriate expression controlelements and a selectable marker. The selectable marker in the vectorconfers resistance to the selection and allows cells to stably integratethe vector into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. A number of selectionsystems may be used, including but not limited to the herpes simplexvirus thymidine kinase (Wigler et al., 1977, Cell 11: 223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection byusing, for example, dhfr, which confers resistance to methotrexate(Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al.,1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confersresistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl.Acad. Sci. USA 78: 2072; neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and/or hyg, which confers resistance to hygromycin (Santerre et al.,1984, Gene 30: 147).

Stable, high yield expression can also be achieved using retroviralvectors that integrate into the host cell genome (see, e.g., U.S. PatentPublications No. 2008/0286779 and 2004/0235173). Alternatively, stable,high yield expression of ApoA-I can be achieved by gene activationmethods, which entail activating expression of and amplifying anendogenous ApoA-I gene in genomic DNA of a mammalian cell of choice, forexample as described in WO 1994/012650. Increasing the copy number of anApoA-I gene (containing an ApoA-I coding sequence and one or morecontrol elements) can facilitate the high yield expression of ApoA-I.Preferably, the mammalian host cell in which ApoA-I is expressed has anApoA-I gene copy index of at least 2, at least 3, at least 4, or atleast 5. In specific embodiments, the mammalian host cell in whichApoA-I is expressed has an ApoA-I gene copy index of at least 6, atleast 7, at least 8, at least 9, or at least 10.

In certain embodiments, the mammalian cells are adapted to produceApoA-I in quantities of at least 0.5 g/L, at least 1 g/L, at least 1.5g/L, at least 2 g/L, at least 2.5 g/L, at least 3 g/L, at least 3.5 g/L,and optionally up to 4 g/L, up to 4.5 g/L, up to 5 g/L, up to 5.5 g/L,or up to 6 g/L. The mammalian host cells are preferably capable ofproducing at least about 0.5, 1, 2, or 3 g/L ApoA-I in culture and/or upto about 20 g/L ApoA-I in culture, e.g., up to 4, 5, 6, 7, 8, 9, 10, 12,or 15 g/L ApoA-I in culture.

In certain embodiments, the mammalian cells are adapted for growth inserum-free medium. In these embodiments, the ApoA-I is secreted from thecells. In other embodiments, the ApoA-I is not secreted from the cells.

The mammalian host cells provided herein can be used to produce ApoA-I.Generally, the methods comprise culturing a mammalian host cell asdescribed herein under conditions in which ApoA-I is expressed.Furthermore, the methods can comprise recovering and, optionally,purifying mature ApoA-I from the supernatant of the mammalian cellculture.

The culture conditions, including the culture medium, temperature, pH,can be suited to the mammalian host cell being cultured and the mode ofculture chosen (shake flask, bioreactor, roller bottle, etc. . . . ).Mammalian cells can be grown in large scale batch culture, in continuousor semi-continuous culture.

Also provided herein is a mammalian cell culture comprising a pluralityof a ApoA-I-producing mammalian host cells described herein. In someembodiments, the mammalian cell culture comprises at least 0.5 g/L, atleast 1 g/L, at least 1.5 g/L, at least 2 g/L, at least 2.5 g/L, atleast 3 g/L, at least 3.5 g/L, and optionally up to 4 g/L, up to 4.5g/L, up to 5 g/L, up to 5.5 g/L, or up to 6 g/L of ApoA-I. The culturecan be of any scale, ranging from about 150 mL to about 500 mL, 1 L, 10L, 15 L, 50 L, 100 L, 200 L, 250 L, 300 L, 350 L, 400 L, 500 L, 750 L,1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 5000 L, 7500 L, 10000 L, 15000L, 20000 L, 25000 L, 50000 L or more. In some instances, the culture isa large scale culture, such as 15 L, 20 L, 25 L, 30 L, 50 L, 100 L, 200L, 300 L, 500 L, 1000 L, 5000 L, 10000 L, 15000 L, 20000 L, 25000 L, upto 50000 L or more.

6.1.3. Purification of Apolipoproteins

The present disclosure relates to methods of obtaining highly purifiedapolipoprotein, that is useful in making lipoprotein complexes andcompositions thereof as described herein. The methods can be applied toany apolipoprotein, including but not limited to, ApoA-I, -II, -III or-IV; ApoB48 and ApoB 100; ApoC-I, -II, -III or -IV; ApoD; ApoE, ApoH;ApoJ. More specifically, the present disclosure relates to methods ofobtaining highly purified ApoA-I. In some embodiments, the ApoA-I is ahuman protein having a sequence selected from, but not limited to, thesequences set forth in Genbank Accession Nos. NP 000030, AAB59514,P02647, CAA30377, AAA51746 and AAH05380.1. In certain embodiments, theApoA-I is a human protein as described above in Section 6.1.2. In otherembodiments, the methods of the present disclosure can be used to purifyApoA-I obtained from non-human animals (see, e.g., U.S. Publication No.2004/0077541), for example, cows, horses, sheep, monkeys, baboons,goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs,hamsters, duck, chicken, salmon and eel (Brouillette et al., 2001,Biochim. Biophys. Acta. 1531:4-46; Yu et al., 1991, Cell Struct. Funct.16(4):347-55; Chen and Albers, 1983, Biochim Biophys Acta. 753(1):40-6;Luo et al., 1989, J Lipid Res. 30(11):1735-46; Blaton et al., 1977,Biochemistry 16:2157-63; Sparrow et al., 1995, J Lipid Res.36(3):485-95; Beaubatie et al., 1986, J. Lipid Res. 27:140-49; Januzziet al., 1992, Genomics 14(4):1081-8; Goulinet and Chapman, 1993, J.Lipid Res. 34(6):943-59; Collet et al., 1997, J Lipid Res. 38(4):634-44;and Frank and Marcel, 2000, J. Lipid Res. 41(6):853-72).

Examples of ApoA-I proteins that can be purified by the methodsdisclosed herein include, but are not limited to, thepreproapolipoprotein form of ApoA-I, pro- and mature forms of ApoA-I,and active polymorphic forms, isoforms, variants and mutants as well astruncated forms, e.g., ApoA-I_(M), ApoA-I_(Z), and ApoA-Ip. ApoA-I_(M)is the R173C molecular variant of ApoA-I (see, e.g., Parolini et al.,2003, J Biol Chem. 278(7):4740-6; Calabresi et al., 1999, Biochemistry38:16307-14; and Calabresi et al., 1997, Biochemistry 36:12428-33).ApoA-I_(P) is the R151C molecular variant of ApoA-I (see, e.g., Daum etal., 1999, J Mol Med. 77(8):614-22). ApoA-I_(L) is an L144R molecularvariant of ApoA-I (see Recalde et al., 2001, Atherosclerosis154(3):613-623; Fiddyment et al., 2011, Protein Expr. Purif.80(1):110-116). Apolipoprotein A-I mutants containing cysteine residuesare also known, and can also be purified by the methods described herein(see, e.g., U.S. Publication No. 2003/0181372). ApoA-I for use in themethods described herein can be in the form of monomers, homodimers, orheterodimers. For example, homo- and heterodimers of pro- and matureApoA-I that can be prepared include, among others, ApoA-I (Duverger etal., 1996, Arterioscler Thromb Vasc Biol. 16(12):1424-29), ApoA-I_(M)(Franceschini et al., 1985, J Biol Chem. 260:1632-35), and ApoA-Ip (Daumet al., 1999, J Mol Med. 77:614-22).

The purification methods described herein can be performed on any scaleconvenient for the skilled practitioner.

In some aspects, ApoA-I protein that can be purified by the methodsdescribed herein has an amino acid sequence that is at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 91% identical, at least 92% identical, at least 93%identical, at least 94% identical, at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, at least 99%identical or at least 100% identical to amino acids 25-267 of SEQ ID NO:1.

Apolipoprotein can be from any source, including from blood plasma orfrom recombinant expression in prokaryotic or eukaryotic cells. Inparticular embodiments, the apolipoprotein is ApoA-I, e.g., humanApoA-I. In some aspects, the ApoA-I is expressed in the cytoplasm orperiplasm of prokaryotic or eukaryotic host cells. In these embodiments,the cells are disrupted to release ApoA-I into the supernatant prior topurifying the ApoA-I. Cell disruption methods are well-known in the art.Exemplary methods of disrupting cells include, but are not limited to,enzymatic methods, sonication, detergent methods, and mechanicalmethods. In certain preferred aspects, ApoA-I is expressed in mammaliancells, preferably CHO cells, and is secreted into the growth medium. Inthese embodiments, ApoA-I is purified from the clarified cell-freemedium. It will be understood that, although the purification methodsare described in detail herein in connection with human ApoA-I, it iswithin the skill in the art to adapt the purification conditions toother apolipoproteins, as well as to non-human ApoA-I, polymorphicforms, isoforms, variants, mutants and truncated forms of ApoA-I orother apolipoproteins, depending on specific protein characteristicsthat are readily ascertainable by the skilled artisan (e.g., molecularweight, isoelectric point, Stokes radius, hydrophobicity, multimericstate, etc.).

Where ApoA-I, is prepared from blood plasma, it can be separated fromblood plasma by any known method, including but not limited to, coldfractionation processes such as those described by Cohn et al., 1946, J.Am. Chem. Soc. 68:459-475 (“Cohn process”) or by Oncley et al., 1949, J.Am. Chem. Soc. 71:541-550 (“Cohn-Oncley process”). Other methods forisolating apolipoprotein from blood plasma include variations of theCohn and Cohn-Oncley processes, such as the Kistler-Nitschmann process.(See Nitschmann et al., 1954, Helv. Chim. Acta 37:866-873; Kistler etal., 1962, Vox Sang. 7:414-424).

In these embodiments, apolipoprotein is obtained by precipitation fromplasma with cold alcohol, e.g., ethanol. Other alcohols for use in coldfractionation of plasma include C1-C6 straight or branched chainalcohols, such as methanol, n-propanol, isopropanol, n-butanol,sec-butanol, isobutanol and tert-butanol. In various embodiments, agentsother than alcohols that reduce protein solubility can be used toprecipitate apolipoprotein from plasma. Such agents include, but are notlimited to, ethers, ammonium sulfate, 7-ethoxyacridine-3,9-diamine(rivanol) and polyethylene glycols. Precipitated proteins can beseparated from the supernatant by any method known in the art,including, but not limited to, sedimentation, centrifugation andfiltration.

ApoA-I can be recovered from any fraction of blood plasma that containsthe protein. In some embodiments, ApoA-I is recovered from a serumfraction of human plasma from which the amount of fibrinogen has beenreduced by precipitation with about 8% (w/w) ethanol. In otherembodiments, apolipoprotein is recovered from a serum fraction of humanplasma from which the concentrations of other serum proteins (e.g.,β-globulins and γ-gamma globulins) have been reduced by precipitationwith about 25% (w/w) ethanol. In still other embodiments, apolipoproteinis recovered as a precipitate from human serum obtained by increasingthe ethanol concentration to about 38% to about 42% (w/w). In aparticular embodiment, apolipoprotein is recovered as a precipitate fromhuman serum obtained by increasing the ethanol concentration to about40% (w/w) (Cohn's fraction IV). Precipitated ApoA-I can be recoveredfrom serum fractions by any method known in the art, including but notlimited to centrifugation and filtration.

In some embodiments, the temperature of plasma fractions from whichapolipoprotein is recovered is sufficiently low to prevent denaturationof the protein. In these embodiments, the temperature of the ApoA-Ifractions ranges from about −10° C. to about 0° C., such as from about−8° C. to about −2° C. In various embodiments, the pH of plasmafractions from which ApoA-I is recovered is in a range that preventsdenaturation of the protein. In these embodiments, the pH of fractionsthat contain ApoA-I ranges from about 5 to about 7, such as from about5.5 to about 6.5.

6.1.4. Improved Lipoprotein Purification Processes

Applicants have further discovered an improved process of purification,described below and illustrated in the Examples, that produceslipoproteins that are mature, intact, and substantially free ofcontaminants. The purification methods described herein can be performedon any scale convenient for the skilled practitioner.

The methods can be applied to any apolipoprotein, including but notlimited to, ApoA-I, -II, -III or -IV; ApoB48 and ApoB 100; ApoC-I, -II,-III or -IV; ApoD; ApoE, ApoH; ApoJ. More specifically, the presentdisclosure relates to methods of obtaining highly purified ApoA-I. Insome embodiments, the ApoA-I is a human protein having a sequenceselected from, but not limited to, the sequences set forth in GenbankAccession Nos. NP 000030, AAB59514, P02647, CAA30377, AAA51746 andAAH05380.1. In certain embodiments, the ApoA-I is a human protein asdescribed above in Section 6.1.2. In other embodiments, the methods ofthe present disclosure can be used to purify ApoA-I obtained fromnon-human animals (see, e.g., U.S. Publication 2004/0077541), forexample, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs,hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, duck,chicken, salmon and eel (Brouillette et al., 2001, Biochim Biophys Acta.1531:4-46; Yu et al., 1991, Cell Struct Funct. 16(4):347-55; Chen andAlbers, 1983, Biochim Biophys Acta. 753(1):40-6; Luo et al., 1989, JLipid Res. 30(11):1735-46; Blaton et al., 1977, Biochemistry 16:2157-63;Sparrow et al., 1995, J Lipid Res. 36(3):485-95; Beaubatie et al., 1986,J Lipid Res. 27:140-49; Januzzi et al., 1992, Genomics 14(4):1081-8;Goulinet and Chapman, 1993, J Lipid Res. 34(6):943-59; Collet et al.,1997, J Lipid Res. 38(4):634-44; and Frank and Marcel, 2000, J LipidRes. 41(6):853-72).

Examples of ApoA-I proteins that can be purified by the methodsdisclosed herein include, but are not limited to, thepreproapolipoprotein form of ApoA-I, pro- and mature forms of ApoA-I,and active polymorphic forms, isoforms, variants and mutants as well astruncated forms, e.g., ApoA-I_(M), ApoA-I_(Z), and ApoA-I_(P).Apolipoprotein A-I mutants containing cysteine residues are also known,and can also be purified by the methods described herein (see, e.g.,U.S. Publication 2003/0181372). ApoA-I for use in the methods describedherein can be in the form of monomers, homodimers, or heterodimers. Forexample, homo- and heterodimers of pro- and mature ApoA-I that can beprepared include, among others, ApoA-I (Duverger et al., 1996,Arterioscler Thromb Vasc Biol. 16(12):1424-29), ApoA-I_(M) (Franceschiniet al., 1985, J Biol Chem. 260:1632-35), and ApoA-I_(P) (Daum et al.,1999, J Mol Med. 77:614-22).

In some aspects, ApoA-I protein that can be purified by the methodsdescribed herein has an amino acid sequence that is at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 91% identical, at least 92% identical, at least 93%identical, at least 94% identical, at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, at least 99%identical or at least 100% identical to amino acids 25-267 of SEQ ID NO:1.

Apolipoprotein can be from any source, including from blood plasma orfrom recombinant expression in prokaryotic or eukaryotic cells. Inparticular embodiments, the apolipoprotein is ApoA-I, e.g., humanApoA-I. In some aspects, the ApoA-I is expressed in the cytoplasm orperiplasm of prokaryotic or eukaryotic host cells. In these embodiments,the cells are disrupted to release ApoA-I into the supernatant prior topurifying the ApoA-I. Cell disruption methods are well-known in the art.Exemplary methods of disrupting cells include, but are not limited to,enzymatic methods, sonication, detergent methods, and mechanicalmethods. In certain preferred aspects, ApoA-I is expressed in mammaliancells, preferably CHO cells, and is secreted into the growth medium. Inthese embodiments, ApoA-I is purified from the clarified cell-freemedium. It will be understood that, although the purification methodsare described in detail herein in connection with human ApoA-I, it iswithin the skill in the art to adapt the purification conditions toother apolipoproteins, as well as to non-human ApoA-I, polymorphicforms, isoforms, variants, mutants and truncated forms of ApoA-I orother apolipoproteins, depending on specific protein characteristicsthat are readily ascertainable by the skilled artisan (e.g., molecularweight, isoelectric point, Stokes radius, hydrophobicity, multimericstate, etc.).

Generally, the purification methods comprise the steps of: (a)contacting an ApoA-I containing solution with an anion exchange matrixunder conditions such that the ApoA-I does not bind to the matrix; (b)filtering the ApoA-I containing solution obtained in step (a) through amembrane having a pore size sufficient to remove viruses or viralparticles; (c) passing the filtrate obtained in step (b) through a firstreverse phase chromatography column under conditions such that theApoA-I binds to the matrix; (d) eluting from the first reverse phasechromatography matrix a first ApoA-I containing reverse phase eluateusing a gradient of increasing concentrations of an organic solvent; (e)passing the first ApoA-I reverse phase eluate from step (d) through asecond reverse phase chromatography column under conditions such thatthe ApoA-I binds to the matrix; and (f) eluting from the second reversephase chromatography matrix a second ApoA-I containing reverse phaseeluate using a gradient of increasing concentrations of an organicsolvent. The order in which the steps are performed is not critical. Aswill be apparent to the person of skill in the art, a variety ofpermutations in the order of the steps are possible, some of which aredescribed below.

In certain aspects, the ApoA-I containing solution is conditioned beforecontacting it with an anion exchange matrix in step (a). Conditioning isperformed to adjust the pH of the protein solution so that it is in arange where ApoA-I does not bind to the anion exchange matrix in step(a) (i.e, the protein does not have a net negative charge and step (a)is run in negative mode). In these aspects, the pH of the ApoA-Icontaining solution is from about 5 to about 7, preferably from about5.0 to about 5.6. In particular aspects, the pH is from about 5.1 toabout 5.5. In still other aspects, the pH is about 5.3. Adjustments inpH can be performed by adding an appropriate acid (e.g., hydrochloricacid) or base (e.g., sodium hydroxide) until a pH within the desiredrange is obtained. In some embodiments, the ApoA-I containing solutionis filtered before the conditioning step to remove cells and celldebris. In other embodiments, when a conditioning step is absent, theApoA-I containing solution is optionally filtered before step (a) toremove cells and cell debris.

In some embodiments, step (a) of contacting an ApoA-I containingsolution with an anion exchange matrix is performed by passing theprotein solution through a chromatography column. In these embodiments,the column is packed at a bed height of from about 10 cm to about 50 cm,preferably from about 10 cm to about 30 cm, and more preferably at a bedheight of about 20 cm. In certain aspects, the column is loaded with aprotein solution comprising from about 10 g to about 50 g, such as fromabout 10 g to about 30 g, such as from about 25 g to about 35 g ofApoA-I per liter. In particular embodiments, the column is loaded with aprotein solution comprising up to about 32 g of ApoA-I per liter. Inother embodiments, step (a) is performed in batch mode, i.e., by addingan anion exchange matrix to a protein solution in a flask, mixing for aperiod of time sufficient for binding contaminants to the matrix, andthen separating the matrix material from the protein solution, e.g., byfiltration or centrifugation. In certain embodiments, the proteinsolution is filtered to remove particulates in the solution prior tocontacting it with the anion exchange matrix.

Anion exchange matrices for use in step (a) of the methods describedherein can be any anion exchange matrix known in the art. Suitable anionexchange matrices include, but are not limited to, Q-Sepharose FF,Q-Spherosil, DEAE-Sepharose FF, Q-Cellulose, DEAE-Cellulose andQ-Spherodex. In a particular embodiment, the anion exchange matrix isQ-Sepharose FF (GE Healthcare). In certain aspects, before contactingthe protein solution in step (a) with an anion exchange matrix, thematrix is equilibrated in a buffer having a pH within the preferredranges discussed above such that the ApoA-I does not bind to the matrix.Buffers useful for equilibrating anion exchange matrices prior to step(a) and for performing step (a) are known to the skilled artisan. Inparticular embodiments, the buffer is TAMP A (20 mM sodium phosphate, pH5.3).

In various embodiments, step (a) is used to purify ApoA-I with respectto proteins other than ApoA-I (e.g., host cell proteins), host cell DNAand endotoxin, which bind to the anion exchange matrix and are therebyseparated from ApoA-I, which does not bind to matrix under the pH andsalt conditions described above. In some aspects, at least 75%, at least80%, at least 85%, or at least 90% or more of the amount of ApoA-I inthe starting solution is recovered from the anion exchange step.

In various embodiments, the purification methods comprise a step (b) inwhich the ApoA-I solution from step (a) is filtered using a filter witha pore size that is sufficient to trap viruses and viral particles.Optionally, the step (b) of filtering through a membrane to removeviruses or viral particles is performed after step (f) above, ratherthan after step (a). In certain aspects, the pH of the eluate from theanion exchange matrix is adjusted before viral filtration step (b) bythe addition of sodium hydroxide or other suitable base. The ApoA-Icontaining solution from step (a) is adjusted to a pH of from about 7.8to about 8.2. In a particular aspect, the ApoA-I containing solution isadjusted to a pH of about 8.0. The filter used in step (b) can be anyfilter with an appropriate pore size for trapping viruses, e.g., with apore size of from about 15 nm to about 75 nm. In particular embodiments,the pore size of the filter is about 20 nm (e.g., Planova 20N, AsahiKasei Medical). The skilled artisan will appreciate that the flow rateof the protein solution through the viral filter is determined by theproperties of the solution (e.g., its viscosity, the concentration ofparticulates, etc.). A typical flow rate for viral filtration is about12.5 L/h/m2, however, the flow rate can be adjusted higher or lower tomaintain a filter pressure of 1 bar or less. The filtrate from step (b)contains ApoA-I. In certain aspects, recovery of ApoA-I from the viralfiltration step is at least 80%, at least 85%, at least 90%, at least95% or at least 98% or more of the amount of the ApoA-I in the anionexchange eluate from step (a).

In particular embodiments, the purification methods described hereincomprise a step (c) after step (b) in which the filtrate from step (b)is passed through a first reverse phase chromatography column underbuffer and salt conditions that allow the Apolipoprotein A-I to bind tothe matrix. In these embodiments, the ApoA-I is purified with respect tohost cell DNA, host cell proteins, endotoxin and truncated forms using agradient of increasing concentrations of organic solvent. Reverse phasechromatography can be performed using a wide variant of matrices knownin the art, including but not limited to silica, polystyrene, orcrosslinked agarose based media onto which C4 to C18 hydrophobic ligandsare grafted. Commercially available hydrophobic matrices useful in themethods described herein include, but are not limited to ButylSepharose-FF, Octyl Sepharose-FF, Dianon HP20ss, C18 Hypersil and Source30 RPC. In particular embodiments, the matrix used in step (c) is Source30 RPC (GE Healthcare). In certain aspects, the reverse phasechromatography column has a bed height of from about 10 cm to about 50cm, such as from about 10 cm to about 30 cm. In a particular aspect, thereverse phase chromatography column has a bed height of about 25 cm.

In some embodiments, the ApoA-I filtrate from the viral filtration step(b) is loaded onto the reverse phase column at a concentration of about1 to about 20 g ApoA-I, such as at a concentration of about 1.5 g toabout 5 g of ApoA-I, and more preferably at a concentration of about 2.5g to about 3.5 g ApoA-I per liter. In a particular aspect, the ApoA-Ifiltrate from step (b) is loaded onto the reverse phase column at aconcentration of about 3.4 g of ApoA-I per liter. Buffer conditions thatcan be used to equilibrate the reverse phase column before loadingApoA-I and to insure that the protein will bind to the column uponloading will be readily ascertainable to those of skill in the art.Preferably, the column equilibration buffer is a strong buffer that canreduce the column pH to about 9.5. In certain embodiments, theequilibration buffer is TAMP D (20 mM ammonium carbonate). Preferably,after equilibration, ApoA-I containing filtrate from step (b) (at a pHof about 8.0) is loaded onto the column at a flow rate of about 0.5 cmto about 5.0 cm per minute, such as from about 2.0 cm to about 4.0 cmper minute. In a particular embodiment, the ApoA-I containing filtrateis loaded onto the column at a flow rate of about 2.8 cm per minute.

After ApoA-I is bound to the reverse phase matrix in step (c), theprotein is eluted in step (d) by exposure to a gradient of increasingconcentrations of organic solvent in buffer, such as from about 35% toabout 50% acetonitrile in TAMP D buffer. In some aspects, a lineargradient of from about 35% to about 50% acetonitrile over a period ofabout 60 to about 90 minutes, such as about 70 minutes or morepreferably about 80 minutes can be used to elute the ApoA-I from thecolumn. In certain aspects, the linear gradient is followed by about 10minutes of isocratic elution with 50% acetonitrile. The exact conditionsfor eluting ApoA-I from the reverse phase column will be readilyascertainable to the skilled artisan. In various embodiments, about 60%,such as about 65%, about 70%, about 75% or about 80% or more of ApoA-Iin the column load is present in the column eluate in step (d).

In certain aspects, the purification methods described herein furthercomprise after step (d) a step (e) of passing the Apolipoprotein A-Ireverse phase eluate from step (d) through a second reverse phasechromatography column in order to further remove DNA, host cell proteinsand truncated forms of ApoA-I from the full-length protein. Preferably,the reverse phase eluate from step (d) is loaded on the second reversephase column under conditions that allow the Apolipoprotein A-I to bindto the matrix. The reverse phase matrix for use in step (e) can be thesame type of matrix or a different type of matrix as used in step (d).In particular embodiments, the reverse phase matrix used in step (e) isa C18 silica matrix, such as a Daisogel SP-300-BIO C18 matrix (300 A, 10μM; Daiso Co., Ltd.). Buffer conditions that can be used to equilibratethe C18 column before loading ApoA-I and to insure that the protein willbind to the column upon loading will be readily ascertainable to thoseof skill in the art. Preferably, the column equilibration buffer is astrong buffer that can reduce the column pH to about 9.5. In certainembodiments, the equilibration buffer is TAMP E (100 mM ammoniumcarbonate). In various embodiments, the reverse phase column used instep (e) of the purification methods described herein has a bed heightof from about 10 cm to about 50 cm, such as from about 10 cm to about 30cm. In particular embodiments, the reverse phase column used in step (e)has a bed height of about 25 cm.

In various embodiments, the ApoA-I eluate from step (d) is loaded ontothe C18 reverse phase column at a concentration of about 0.5 g to about30 g ApoA-I, such as at a concentration of about 1 g to about 10 g ofApoA-I, and more preferably at a concentration of about 4 g to about 5 gof ApoA-I per liter. In particular embodiments, the ApoA-I eluate fromstep (d) is loaded onto the C18 column at a concentration of about 4.7 gof ApoA-I per liter. Preferably, after equilibration, ApoA-I containingeluate from step (d) is loaded onto the column at a flow rate of fromabout 0.5 cm to about 5.0 cm per minute, such as from about 1.0 cm toabout 3.0 cm per minute. In a particular embodiment, the ApoA-Icontaining filtrate is loaded onto the column at a flow rate of about2.1 cm per minute.

After ApoA-I is bound to the reverse phase matrix in step (e), theprotein is eluted in step (f) using a gradient of increasingconcentrations of organic solvent in buffer, such as from about 40% toabout 50% acetonitrile in TAMP E buffer. In some aspects, a lineargradient of from about 40% to about 50% acetonitrile is used to elutethe ApoA-I from the column over a period of about 40 to about 80minutes, such as about 50 minutes, about 60 minutes or about 70 minutes.In particular embodiments, a linear gradient of from about 40% to about50% acetonitrile in TAMP E buffer over a period of about 60 minutes isused to elute the ApoA-I from the reverse phase matrix. In certainaspects, the linear gradient is followed by about 15 minutes ofisocratic elution with 50% acetonitrile. The exact conditions foreluting ApoA-I from the reverse phase column will be readilyascertainable to the skilled artisan. In various embodiments, about 60%,such as about 65%, about 70%, about 75% or about 80% or more of ApoA-Iin the column load is recovered in the column eluate in step (f).

In certain embodiments, the organic solvent is removed from the ApoA-Icontaining eluate obtained in step (f) of the methods described herein.Solvent removal can be accomplished by any method known in the art,including, but not limited to, concentrating the ApoA-I containingeluate obtained in step (f) and diafiltering the concentrate into anaqueous buffer. In certain embodiments, the eluate from step (f) isconcentrated by about 2-fold, by about 2.5-fold, by about 3-fold, byabout 3.5-fold, by about 4-fold, by about 4.5-fold or by about 5-fold ascompared to the volume of eluate from the reverse phase column in step(f). In a particular embodiment, the eluate is concentrated by about2.5-fold and is then diafiltered against approximately 10, 15, or 20,preferably 15 volumes of a suitable aqueous buffer. Suitable aqueousbuffers are known in the art. A particularly preferred buffer is TAMP C(3 mM sodium phosphate, pH 8.0).

In some embodiments the order of chromatography columns is reversed.

Optionally, after concentration and buffer exchange, the aqueous ApoA-Isolution is further purified by anion exchange chromatography innegative mode (i.e., under conditions where the ApoA-I does not bind tothe anion exchange matrix) to remove residual DNA and other negativelycharged contaminants such as host cell proteins. (Step (g)). In someembodiments, the anion exchange step is performed in batch mode. Inother embodiments, the anion exchange step is performed by columnchromatography. Suitable anion exchange matrices for use in batch modeor in column chromatography include, but are not limited to, QSepharose-FF or any of the anion exchange matrices discussed above foruse in step (a). In particular aspects, the anion exchange step isperformed by passing the ApoA-I solution through an anion exchangemembrane, such as a membrane having a large surface-area and a strongcationic charge, e.g., Sartobind Q or Mustang Q. Preferably, the anionexchange step is performed using a Mustang Q anion exchange membrane(Pall Life Sciences). In certain aspects, the pH of the aqueous ApoA-Isolution is reduced to about 5.5, to about 6.0 or to about 6.5 beforethis anion exchange step using any suitable acid. In particular aspects,the pH of the aqueous ApoA-I solution is reduced to about 6.0 usingdilute phosphoric acid. In preferred embodiments, the ApoA-I solution ispassed through a Mustang Q cartridge at approximately 12.5 L/m2/h.

In some embodiments, the anion exchange membrane filtrate isconcentrated and optionally dialfiltered to exchange the solvent to onethat is suitable for storage or for further processing of the ApoA-I,such as complexing with lipids as described below in Section 6.5.1and/or formulation in pharmaceutical compositions as described below inSection 6.6. Suitable buffers for storage or further processing ofApoA-I are readily ascertainable to the skilled artisan. In particularembodiments, the purified ApoA-I is exchanged into TAMP C buffer. Anyultrafiltration membrane can be used in this step, provided that themembrane has a molecular weight cutoff that is below the molecularweight of full-length mature ApoA-I such that it allows the passage ofbuffer but not protein. In particular embodiments, a polyethersulphonemembrane (e.g., Filtron Omega series) of 10,000 nominal molecular weightcut-off is used. Preferably, the ApoA-I concentration in the solutionafter ultrafiltration is at least 10 g/L, at least 12 g/L, at least 15g/L, at least 20 g/L, at least 25 g/L, at least 30 g/L, at least 35 g/L,at least 40 g/L, at least 45 g/L or at least 50 g/L.

6.1.5. Apolipoprotein Products

The present disclosure also provides substantially pure maturefull-length apolipoproteins. As used herein, the term “substantiallypure” refers to a protein that is at least 95% pure. In certainembodiments, the substantially pure protein is at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100%pure. In certain aspects, the substantially pure apolipoprotein productproduced by the purification methods described herein is a clear toslightly opalescent colorless solution free of visible particles whenvisually inspected using a light source against a white background. Invarious embodiments, substantially pure apolipoprotein obtained orobtainable by the methods described in Section 6.1.4 above, compriseslow or undetectable amounts of one or more of host cell DNA, proteinsother than the apolipoprotein (e.g., host cell proteins), endotoxin,residual solvent, as well as low bioburden (i.e., low number of microbeson or in the sample), as described in further detail below. The purityof the apolipoprotein product can be determined by any method known inthe art, including, but not limited to, N-terminal Edman sequencing,MALDI-MS, gel electrophoresis, HPLC, and/or immunoassay.

In various embodiments, the substantially pure apolipoprotein productobtained by the methods described herein is full-length mature humanApoA-I having a mass that is about 28.1 kilodaltons. The mass of ApoA-Iin the product can be determined by any method known in the art,including, but not limited to, MALDI-MS. In various embodiments, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% of the ApoA-I protein in theproduct is mature full-length ApoA-I (e.g., ApoA-I comprising aminoacids 25 to 267 of SEQ ID NO: 1). In certain aspects, the substantiallypure ApoA-I product comprises about 15% or less, about 10% or less,about 5% or less, about 4% or less, about 3% or less, about 2% or lessor about 1% or less by weight of N-terminally extended ApoA-I isoforms(e.g., proApoA-I). As will be appreciated by the skilled artisan, anyN-terminally extended ApoA-I in the product will be rapidly converted tomature ApoA-I in the blood upon administration. In various embodiments,the ApoA-I product comprises about 25% or less, about 20% or less, about15% or less, about 10% or less, about 5% or less, about 4% or less,about 3% or less, about 2% or less, about 1% or less, about 0.75% orless, about 0.5% or less, about 0.25% or less, or about 0.1% or less byweight of truncated forms of ApoA-I. The amount of truncated or extendedApoA-I can be determined, for example, by N-terminal Edman sequencingand/or MALDI-MS and/or by running and scanning an SDS-PAGE gel todetermine the ratio of the intensity of the purified ApoA-I band area tothe total intensity of all bands, if present. In various embodiments,the ApoA-I product comprises about 20% or less, about 10% or less, about5% or less, about 4% or less, about 3% or less, about 2% or less, about1% or less, about 0.75% or less, about 0.5% or less, about 0.25% orless, or about 0.1% or less by weight of oxidized forms of ApoA-I, inparticular ApoA-I oxidized at position Met₁₁₂ and/or Met₁₄₈.

In certain embodiments, the substantially pure apolipoprotein producedby the methods described herein comprises host cell proteins in anamount that is less than about 100 ppm (e.g., ng/mg), such as less thanabout 75 ppm, less than about 50 ppm, less than about 40 ppm, less thanabout 30 ppm, less than about 20 ppm, or less than about 10 ppm. Inparticular embodiments, the substantially pure apolipoprotein productcomprises less than about 20 ppm of host cell proteins. More preferably,the apolipoprotein product comprises less than about 10 ppm of host cellproteins. The presence and amount of host cell proteins in anapolipoprotein sample can be determined by any method known in the art.When apolipoprotein is produced recombinantly in, e.g., mammalian cells,commercially available ELISA kits (e.g., Kit F015 from CygnusTechnologies) can be used to detect and quantitate levels of host cellproteins.

In some aspects, the substantially pure apolipoprotein product purifiedas described herein comprises host cell DNA in an amount that is lessthan about 50 pg/mg of apolipoprotein, such as less than about 40 pg/mg,less than about 30 pg/mg, less than about 20 pg/mg, less than about 10pg/mg, or less than about 5 pg/mg of apolipoprotein. In preferredembodiments, the substantially pure apolipoprotein product comprisesless than about 10 pg of host cell proteins per mg of apolipoprotein.The presence and amount of host cell DNA in an apolipoprotein sample canbe determined by any method known in the art, including real time-PCR ordetection of complexes with single stranded binding protein using ananti-SSB antibody (Glycotype Biotechnology), preferably by quantitativePCR.

In certain embodiments, the substantially pure apolipoprotein productproduced by the methods described herein comprises endotoxin in anamount that is less than about 0.5 EU per mg of apolipoprotein, such asless than about 0.4 EU per mg, less than about 0.3 EU per mg, less thanabout 0.2 EU per mg or less than about 0.1 EU per mg of apolipoprotein.Preferably, the substantially pure apolipoprotein product describedherein comprises less than about 0.1 EU of endotoxin per mg ofapolipoprotein. Detection and quantitation of endotoxin can be achievedby any method known in the art, for example using the Limulus AmebocyteLysate (LAL) qualitative test for gram-negative bacterial endotoxins.(Cambrex; sensitivity 0.125 EU/mL).

The substantially pure apolipoprotein product described herein has a lowbioburden. The term “bioburden” refers to the level of aerobic bacteria,anaerobic bacteria, yeast and molds in the product. In variousembodiments, the bioburden of the substantially pure apolipoproteinproduct purified as described herein is less than about 1 CFU per mL.Bioburden testing can be performed according to any known method, forexample according to the European Pharmacopoeia Chapter 2.6.12.B, 2.6.1and USB Chapter 61 harmonized method.

The substantially pure apolipoprotein product described herein compriseslow amounts of residual solvents. In particular embodiments, residualsolvent is present in an amount that is less than about 50 ppm, lessthan about 45 ppm, less than about 40 ppm, less than about 35 ppm, lessthan about 30 ppm, less than about 25 ppm, less than about 20 ppm, lessthan about 15 ppm or less than about 10 ppm for 10 mg/L ofapolipoprotein. Preferably, residual solvent is present at an amountthat is less than about 41 ppm for 10 mg/L of apolipoprotein. The amountof residual solvent can be assayed by any method known in the art,including, but not limited to, GC-MS and HPLC.

Preferably, the apolipoprotein is ApoA-I (e.g., ApoA-I comprising aminoacids 25-267 of SEQ ID NO: 1). In some embodiments, the mature humanApoA-I protein has an amino acid sequence having an aspartic acid atposition 1 (i.e., the position corresponding to position 25 of SEQ IDNO:1).

6.2. The Lipid Fraction

The lipoprotein complexes and compositions of the present disclosurecomprise a lipid fraction. The lipid fraction includes one or morelipids. In various embodiments, one or more lipids can be saturatedand/or unsaturated, and natural or synthetic lipids. The lipid fractionpreferably includes at least one phospholipid.

Suitable lipids that can be present in the lipid fraction include, butare not limited to, small alkyl chain phospholipids, eggphosphatidylcholine, soybean phosphatidylcholine,dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholinc,distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerolssuch as dimyristoylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid,dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, palmitoylsphingomyelin, dipalmitoylsphingomyelin, eggsphingomyelin, milk sphingomyelin, phytosphingomyelin,distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives. Synthetic lipids, suchas synthetic palmitoylsphingomyelin orN-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form ofphytosphingomyelin) can be used to minimize lipid oxidation.Phospholipid fractions including palmitoylsphingomyelin can optionallyinclude small quantities of any type of lipid, including but not limitedto lysophospholipids, sphingomyelins other than palmitoylsphingomyelin,galactocerebroside, gangliosides, cerebrosides, glycerides,triglycerides, and cholesterol and its derivatives.

In preferred embodiments, the lipid fraction includes two types ofphospholipids: a sphingomyelin (SM) and a negatively chargedphospholipid. SM is a “neutral” phospholipid in that it has a net chargeof about zero at physiological pH. The identity of the SM used is notcritical for success. Thus, as used herein, the expression “SM” includessphingomyelins derived or obtained from natural sources, as well asanalogs and derivatives of naturally occurring SMs that are imperviousto hydrolysis by LCAT, as is naturally occurring SM. SM is aphospholipid very similar in structure to lecithin, but, unlikelecithin, it does not have a glycerol backbone, and hence does not haveester linkages attaching the acyl chains. Rather, SM has a ceramidebackbone, with amide linkages connecting the acyl chains. SM is not asubstrate for LCAT, and generally cannot be hydrolyzed by it. It canact, however, as an inhibitor of LCAT or can decrease LCAT activity bydiluting the concentration of the substrate phospholipid. Because SM isnot hydrolyzed, it remains in the circulation longer. It is expectedthat this feature will permit the negatively charged lipoproteincomplexes described herein to have a longer duration of pharmacologicaleffect (mobilization of cholesterol) and to pick up more lipids, inparticular cholesterol, than apolipoprotein complexes that do notinclude SM. This effect may result in less frequent or smaller dosesbeing necessary for treatment than are required for lipoproteincomplexes that do not include SM.

The SM may be obtained from virtually any source. For example, the SMmay be obtained from milk, egg or brain. SM analogues or derivatives mayalso be used. Non-limiting examples of useful SM analogues andderivatives include, but are not limited to, palmitoylsphingomyelin,N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form ofphytosphingomyelin), palmitoylsphingomyelin, stearoylsphingomyelin,D-erythro-N-16:0-sphingomyelin and its dihydro isomer,D-erythro-N-16:0-dihydro-sphingomyelin. Synthetic SM such as syntheticpalmitoylsphingomyelin orN-palmitoyl-4-hydroxysphinganine-1-phosphocholine (phytosphingomyelin)can be used in order to produce more homogeneous complexes and withfewer contaminants and/or oxidation products than sphingolipids ofanimal origin.

Exemplary sphingomyelins palmitoylsphingomyelin and phytosphingomyelinare shown below.

Sphingomyelins isolated from natural sources may be artificiallyenriched in one particular saturated or unsaturated acyl chain. Forexample, milk sphingomyelin (Avanti Phospholipid, Alabaster, Ala.) ischaracterized by long saturated acyl chains (i.e., acyl chains having 20or more carbon atoms). In contrast, egg sphingomyelin is characterizedby short saturated acyl chains (i.e., acyl chains having fewer than 20carbon atoms). For example, whereas only about 20% of milk sphingomyelincomprises C16:0 (16 carbon, saturated) acyl chains, about 80% of eggsphingomyelin comprises C16:0 acyl chains. Using solvent extraction, thecomposition of milk sphingomyelin can be enriched to have an acyl chaincomposition comparable to that of egg sphingomyelin, or vice versa.

The SM may be semi-synthetic such that it has particular acyl chains.For example, milk sphingomyelin can be first purified from milk, thenone particular acyl chain, e.g., the C16:0 acyl chain, can be cleavedand replaced by another acyl chain. The SM can also be entirelysynthesized, by e.g., large-scale synthesis. See, e.g., Dong et al.,U.S. Pat. No. 5,220,043, entitled Synthesis of D-erythro-sphingomyelins,issued Jun. 15, 1993; Weis, 1999, Chem. Phys. Lipids 102 (1-2):3-12.

The lengths and saturation levels of the acyl chains comprising asemi-synthetic or a synthetic SM can be selectively varied. The acylchains can be saturated or unsaturated, and can contain from about 6 toabout 24 carbon atoms. Each chain may contain the same number of carbonatoms or, alternatively each chain may contain different numbers ofcarbon atoms. In some embodiments, the semi-synthetic or synthetic SMcomprises mixed acyl chains such that one chain is saturated and onechain is unsaturated. In such mixed acyl chain SMs, the chain lengthscan be the same or different. In other embodiments, the acyl chains ofthe semi-synthetic or synthetic SM are either both saturated or bothunsaturated. Again, the chains may contain the same or different numbersof carbon atoms. In some embodiments, both acyl chains comprising thesemi-synthetic or synthetic SM are identical. In a specific embodiment,the chains correspond to the acyl chains of a naturally-occurring fattyacid, such as for example oleic, palmitic or stearic acid. In anotherembodiment, SM with saturated or unsaturated functionalized chains isused. In another specific embodiment, both acyl chains are saturated andcontain from 6 to 24 carbon atoms. Non-limiting examples of acyl chainspresent in commonly occurring fatty acids that can be included insemi-synthetic and synthetic SMs are provided in Table 1, below:

TABLE 1 Length:Number of Unsaturations Common Name 14:0 myristic acid16:0 palmitic acid 18:0 stearic acid 18:1 cisΔ⁹ oleic acid 18:2cisΔ^(9, 12) linoleic acid 18:3 cisΔ^(9, 12, 15) linonenic acid 20:4cisΔ^(5, 8, 11, 14) arachidonic acid 20:5 cisΔ^(5, 8, 11, 14, 17)eicosapentaenoic acid (an omega-3 fatty acid)

In preferred embodiments, the SM is palmitoyl SM, such as syntheticpalmitoyl SM, which has C16:0 acyl chains, or is egg SM, which includesas a principal component palmitoyl SM.

In a specific embodiment, functionalized SM, such as aphytosphingomyelin, is used.

The lipid fraction preferably includes a negatively chargedphospholipid. As used herein, “negatively charged phospholipids” arephospholipids that have a net negative charge at physiological pH. Thenegatively charged phospholipid may comprise a single type of negativelycharged phospholipid, or a mixture of two or more different, negativelycharged, phospholipids. In some embodiments, the charged phospholipidsare negatively charged glycerophospholipids. The identity(ies) of thecharged phospholipids(s) are not critical for success. Specific examplesof suitable negatively charged phospholipids include, but are notlimited to, a 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], aphosphatidylglycerol, a phospatidylinositol, a phosphatidylserine, and aphosphatidic acid. In some embodiments, the negatively chargedphospholipid comprises one or more of phosphatidylinositol,phosphatidylserine, phosphatidylglycerol and/or phosphatidic acid. In aspecific embodiment, the negatively charged phospholipid consists of1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], or DPPG.

Like the SM, the negatively charged phospholipids can be obtained fromnatural sources or prepared by chemical synthesis. In embodimentsemploying synthetic negatively charged phospholipids, the identities ofthe acyl chains can be selectively varied, as discussed above inconnection with SM. In some embodiments of the negatively chargedlipoprotein complexes described herein, both acyl chains on thenegatively charged phospholipids are identical. In some embodiments, theacyl chains on the SM and the negatively charged phospholipids are allidentical. In a specific embodiment, the negatively chargedphospholipid(s), and/or SM all have C16:0 or C16:1 acyl chains. In aspecific embodiment the fatty acid moiety of the SM is predominantlyC16:1 palmitoyl. In one specific embodiment, the acyl chains of thecharged phospholipid(s) and/or SM correspond to the acyl chain ofpalmitic acid.

The phospholipids used are preferably at least 95% pure, and/or havereduced levels of oxidative agents. Lipids obtained from natural sourcespreferably have fewer polyunsaturated fatty acid moieties and/or fattyacid moieties that are not susceptible to oxidation. The level ofoxidation in a sample can be determined using an iodometric method,which provides a peroxide value, expressed in milli-equivalent number ofisolated iodines per kg of sample, abbreviated meq O/kg. See, e.g.,Gray, J. I., Measurement of Lipid Oxidation: A Review, Journal of theAmerican Oil Chemists Society, Vol. 55, p. 539-545 (1978); Heaton, F. W.and Uri N., Improved Iodometric Methods for the Determination of LipidPeroxides, Journal of the Science of food and Agriculture, vol 9. P,781-786 (1958). Preferably, the level of oxidation, or peroxide level,is low, e.g., less than 5 meq O/kg, less than 4 meq O/kg, less than 3meq O/kg, or less than 2 meq O/kg.

Lipid fractions including SM and palmitoylsphingomyelin can optionallyinclude small quantities of additional lipids. Virtually any type oflipids may be used, including, but not limited to, lysophospholipids,galactocerebroside, gangliosides, cerebrosides, glycerides,triglycerides, and cholesterol and its derivatives.

When included, such optional lipids will typically comprise less thanabout 15 wt % of the lipid fraction, although in some instances moreoptional lipids could be included. In some embodiments, the optionallipids comprise less than about 10 wt %, less than about 5 wt %, or lessthan about 2 wt %. In some embodiments, the lipid fraction does notinclude optional lipids.

In a specific embodiment, the phospholipid fraction contains egg SM orpalmitoyl SM or phytosphingomyelin and DPPG in a weight ratio(SM:negatively charged phospholipid) ranging from 90:10 to 99:1, morepreferably ranging from 95:5 to 98:2. In one embodiment, the weightratio is 97:3.

The lipoprotein complexes of the present disclosure can also be used ascarriers to deliver hydrophobic, lipophilic or apolar active agents fora variety of therapeutic or diagnostic applications. For suchapplications, the lipid fraction can further include one or morehydrophobic, lipophilic or apolar active agents, including but notlimited to fatty acids, drugs, nucleic acids, vitamins, and/ornutrients. Suitable hydrophobic, lipophilic or apolar active agents arenot limited by therapeutic category, and can be, for example,analgesics, anti-inflammatory agents, antihelmimthics, anti-arrhythmicagents, anti-bacterial agents, anti-viral agents, anti-coagulants,anti-depressants, anti-diabetics, anti-epileptics, anti-fungal agents,anti-gout agents, anti-hypertensive agents, anti-malarial, anti-migraineagents, anti-muscarinic agents, anti-neoplastic agents, erectiledysfunction improvement agents, immunosuppressants, anti-protozoalagents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics,neuroleptics, β-blockers, cardiac inotropic agents, corticosteroids,diuretics, anti-parkinsonian agents, gastro-intestinal agents, histaminereceptor antagonists, keratolytics, lipid regulating agents,anti-anginal agents, cox-2 inhibitors, leukotriene inhibitors,macrolides, muscle relaxants, nutritional agents, nucleic acids (e.g.,small interfering RNAs), opioid analgesics, protease inhibitors, sexhormones, stimulants, muscle relaxants, anti-osteoporosis agents,anti-obesity agents, cognition enhancers, anti-urinary incontinenceagents, nutritional oils, anti-benign prostate hypertrophy agents,essential fatty acids, non-essential fatty acids, and mixtures thereof.

Specific, non-limiting examples of suitable hydrophobic, lipophilic, orapolar active agents are: acetretin, albendazole, albuterol,aminoglutethimide, amiodarone, amlodipine, amphetamine, amphotericin B,atorvastatin, atovaquone, azithromycin, baclofen, beclomethasone,benezepril, benzonatate, betamethasone, bicalutanide, budesonide,bupropion, busulfan, butenafine, calcifediol, calcipotriene, calcitriol,camptothecin, candesartan, capsaicin, carbamezepine, carotenes,celecoxib, cerivastatin, cetirizine, chlorpheniramine, cholecalciferol,cilostazol, cimetidine, cinnarizine, ciprofloxacin, cisapride,clarithromycin, clemastine, clomiphene, clomipramine, clopidogrel,codeine, coenzyme Q10, cyclobenzaprine, cyclosporin, danazol,dantrolene, dexchlorpheniramine, diclofenac, dicoumarol, digoxin,dehydroepiandrosterone, dihydroergotamine, dihydrotachysterol,dirithromycin, donezepil, efavirenz, eposartan, ergocalciferol,ergotamine, essential fatty acid sources, etodolac, etoposide,famotidine, fenofibrate, fentanyl, fexofenadine, finasteride,fluconazole, flurbiprofen, fluvastatin, fosphenytoin, frovatriptan,furazolidone, gabapentin, gemfibrozil, glibenclamide, glipizide,glyburide, glimepiride, griseofulvin, halofantrine, ibuprofen,irbesartan, irinotecan, isosorbide dinitrate, isotretinoin,itraconazole, ivermectin, ketoconazole, ketorolac, lamotrigine,lansoprazole, leflunomide, lisinopril, loperamide, loratadine,lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone,mifepristone, mefloquine, megestrol acetate, methadone, methoxsalen,metronidazole, miconazole, midazolam, miglitol, minoxidil, mitoxantrone,montelukast, nabumetone, nalbuphine, naratriptan, nelfinavir,nifedipine, nilsolidipine, nilutanide, nitrofurantoin, nizatidine,omeprazole, oprevelkin, oestradiol, oxaprozin, paclitaxel, paracalcitol,paroxetine, pentazocine, pioglitazone, pizofetin, pravastatin,prednisolone, probucol, progesterone, pseudoephedrine, pyridostigmine,rabeprazole, raloxifene, rofecoxib, repaglinide, rifabutine,rifapentine, rimexolone, ritanovir, rizatriptan, rosiglitazone,saquinavir, sertraline, sibutramine, sildenafil citrate, simvastatin,sirolimus, spironolactone, sumatriptan, tacrine, tacrolimus, tamoxifen,tamsulosin, targretin, tazarotene, telmisartan, teniposide, terbinafine,terazosin, tetrahydrocannabinol, tiagabine, ticlopidine, tirofibran,tizanidine, topiramate, topotecan, toremifene, tramadol, tretinoin,troglitazone, trovafloxacin, ubidecarenone, valsartan, venlafaxine,verteporfin, vigabatrin, vitamin A, vitamin D, vitamin E, vitamin K,zafirlukast, zileuton, zolmitriptan, zolpidem, and zopiclone. Salts,isomers and derivatives of the above-listed agents may also be used, aswell as mixtures.

6.3. Lipoprotein Complexes

The present disclosure provides lipoprotein complexes comprising aprotein fraction and a lipid fraction, the composition of each of whichhas been described above in Sections 6.1 and 6.2, respectively.

Generally, the protein fraction includes one or more lipid-bindingprotein, such as an apolipoprotein and/or derivative or analog thereofthat provides therapeutic and/or prophylactic benefit. The complexes caninclude a single type of lipid-binding protein, or mixtures of two ormore different lipid-binding proteins, which can be derived from thesame or different species. Suitable lipid-binding proteins are describedabove in Section 6.1. Although not required, the lipoprotein complexeswill preferably comprise lipid-binding proteins that are derived from,or correspond in amino acid sequence to, the animal species beingtreated, in order to avoid inducing an immune response to the therapy.Thus, for treatment of human patients, lipid-binding proteins of humanorigin are preferably used in the complexes of the disclosure. The useof peptide mimetic apolipoproteins can also reduce or avoid an immuneresponse.

The use of apolipoprotein that has a high degree of purity (e.g., matureand not truncated, oxidized, deamidated, contaminated with endotoxinand/or contaminated with other proteins or with nucleic acids) isthought to enhance the therapeutic potency and/or enhance safety oflipoprotein complex. Accordingly, the protein fraction can comprise atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% mature, full-lengthApoA-I, optionally, having no more than 25%, no more than 20%, no morethan 15%, no more than 10%, no more than 5%, or about 0% oxidizedmethionine-112 or methionine-148, and/or no more than 15%, no more than10%, no more than 5%, or about 0% deaminated amino acids. Theapolipoprotein can be purified according to any of the methods describedherein. Preferably, the apolipoprotein can be made as described inSection 6.1.4.

In a specific embodiment, the protein fraction comprises or consistsessentially of ApoA-I, for example, substantially pure mature,full-length ApoA-I, as described above in Section 6.1.5.

The lipid fraction includes one or more lipids, which can be saturated,unsaturated, natural and synthetic lipids and/or phospholipids. Suitablelipids include, but are not limited to, small alkyl chain phospholipids,egg phosphatidylcholine, soybean phosphatidylcholine,dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine,distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerolssuch as dimyristoylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid,dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, egg sphingomyelin, milk sphingomyelin,phytosphingomyelin, palmitoylsphingomyelin, dipalmitoylsphingomyelin,distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives. Phospholipid fractionsincluding SM and palmitoylsphingomyelin can optionally include smallquantities of any type of lipid, including but not limited tolysophospholipids, sphingomyelins other than palmitoylsphingomyelin,galactocerebroside, gangliosides, cerebrosides, glycerides,triglycerides, and cholesterol and its derivatives. Synthetic lipids arepreferred, such as synthetic palmitoyl sphingomyelin orN-palmitoyl-4-hydroxysphinganine-1-phosphocholine (phytosphingomyelin).Further lipids are described above in Section 6.2. Preferably,lipoprotein complexes comprise sphingomyelin.

Optionally, the lipoprotein complexes of the present disclosure can beloaded with hydrophobic, lipophilic or apolar active agents, includingbut not limited to fatty acids, drugs, nucleic acids, vitamins, and/ornutrients, for a variety of therapeutic or diagnostic applications.Suitable agents are described above in Section 6.2.

The lipoprotein complexes can be made using any of the methods describedherein. Preferably, the complexes are made as described in Sections6.5.1 to 6.5.4.

The molar ratio of the lipid fraction to the protein fraction of thenegatively charged lipoprotein complexes described herein can vary, andwill depend upon, among other factors, the identity(ies) of theapolipoprotein comprising the protein fraction, the identities andquantities of the charged phospholipids comprising the lipid fraction,and the desired size of the charged lipoprotein complex. Because thebiological activity of apolipoproteins such as ApoA-I are thought to bemediated by the amphipathic helices comprising the apolipoprotein, it isconvenient to express the apolipoprotein fraction of thelipid:apolipoprotein molar ratio using ApoA-I protein equivalents. It isgenerally accepted that ApoA-I contains 6-10 amphipathic helices,depending upon the method used to calculate the helices. Otherapolipoproteins can be expressed in terms of ApoA-I equivalents basedupon the number of amphipathic helices they contain. For example,ApoA-I_(M), which typically exists as a disulfide-bridged dimer, can beexpressed as 2 ApoA-I equivalents, because each molecule of ApoA-I_(M)contains twice as many amphipathic helices as a molecule of ApoA-I.Conversely, a peptide apolipoprotein that contains a single amphipathichelix can be expressed as a 1/10-1/6 ApoA-I equivalent, because eachmolecule contains 1/10-1/6 as many amphipathic helices as a molecule ofApoA-I. In general, the lipid:ApoA-I equivalent molar ratio of thelipoprotein complexes (defined herein as “Ri”) will range from about105:1 to 110:1. In some embodiments, the Ri is about 108:1. Ratios inweight can be obtained using a MW of approximately 650-800 forphospholipids.

In some embodiments, the molar ratio of lipid:ApoA-I equivalents (“RSM”)ranges from about 80:1 to about 110:1, e.g., about 80:1 to about 100:1.In a specific example, the RSM for lipoprotein complexes can be about82:1.

The various apolipoprotein and/or phospholipids molecules comprising thenegatively charged lipoprotein complexes may be labeled with anyart-known detectable marker, including stable isotopes (e.g., ¹³C, ¹⁵N,²H, etc.); radioactive isotopes (e.g., ¹⁴C, ³H, ¹²⁵I, etc.);fluorophores; chemiluminescers; or enzymatic markers.

In preferred embodiments, the lipoprotein complexes are negativelycharged lipoprotein complexes which comprise a protein fraction which ispreferably mature, full-length ApoA-I, and a lipid fraction comprising aneutral phospholipid, sphingomyelin (SM), and negatively chargedphospholipid.

It has been discovered that composition and relative quantities of SMand negatively charged phospholipid comprising the lipid fraction oflipoprotein complexes affect the homogeneity and stability ofcompositions comprising the complexes. As illustrated in the Examplessection, compositions comprising complexes in which the lipid fractionis composed of SM and negatively charged phospholipid are morehomogeneous, and more stable than similar compositions in which thelipid fraction includes DPPC in addition to SM.

Thus, complexes of the present disclosure that contain SM and negativelycharged lipid are preferably formed in the absence of a lecithin inorder to improve their homogeneity and stability. Once homogeneouscomplexes containing SM and negatively charged lipids are formed,additional lipids such as lecithin can be incorporated.

When included, optional lipids will typically comprise less than about15% of the lipid fraction, although in some instances more optionallipids could be included. In some embodiments, the optional lipidscomprise than about 10%, less than about 5%, or less than about 2% wt %.In some embodiments, the lipid fraction of the negatively chargedlipoprotein complexes does not include optional lipids.

In a specific embodiment, the phospholipid fraction contains eggSM orpalmitoyl SM or phytoSM and DPPG in a weight ratio (SM:negativelycharged phospholipid) ranging from 90:10 to 99:1, more preferablyranging from 95:5 to 98:2, e.g., 97:3.

Some apolipoproteins exchange in vivo from one lipoprotein complex toanother (this is true for apolipoprotein ApoA-I). During the course ofsuch exchange, the apolipoprotein typically carries with it one or morephospholipid molecules. Owing to this property, it is expected that thenegatively charged lipoprotein complexes described herein will “seed”negatively charged phospholipids to endogenous HDL, thereby transformingthem into alpha particles that are more resistant to elimination by thekidneys. Thus, it is expected that administration of the negativelycharged lipoprotein complexes and compositions described herein willincrease serum levels of HDL, and/or alter endogenous HDL half-life aswell as endogenous HDL metabolism. It is expected that this will resultin alteration of cholesterol metabolism and reverse lipid transport.

As illustrated in the Examples section, compositions comprisingcomplexes in which the weight ratio of ApoA-I:SM and DPPG phospholipidis about 1:2.7 were more homogeneous and more stable than similarcompositions with other weight:weight ratios. Accordingly, the presentdisclosure provides lipoprotein compositions in which the protein:lipidweight ratio is optimized for formation of a homogeneous population ofcomplexes. This weight:weight ratio ranges from 1:2.6 to 1:3, and isoptimally 1:2.7, for complexes of ApoA-I, SM, and DPPG, and forcomplexes of components of similar molecular mass. In specificembodiments, the ratio of ApoA-I protein fraction to lipid fractiontypically ranges from about 1:2.7 to about 1:3, with 1:2.7 beingpreferred. This corresponds to molar ratios of ApoA-I protein tophospholipid ranging from approximately 1:90 to 1:140. Accordingly, thepresent disclosure provides complexes in which the molar ratio proteinto lipid is about 1:90 to about 1:120, about 1:100 to about 1:140, orabout 1:95 to about 1:125. Specifically at this optimized ratio, theApoA-I protein fraction and the SM and DPPG lipid fraction formsubstantially homogeneous complexes with the same size and chargecharacteristics, as assayed by column chromatography and gelelectrophoresis, respectively, as natural HDL.

The size of the negatively charged lipoprotein complex can be controlledby varying the Ri. That is, the smaller the Ri, the smaller the disk.For example, large discoidal disks will typically have an Ri in therange of about 200:1 to 100:1, whereas small discoidal disks willtypically have an Ri in the range of about 100:1 to 30:1.

In some specific embodiments, the negatively charged lipoproteincomplexes are large discoidal disks that contain 2-4 ApoA-I equivalents(e.g., 2-4 molecules of ApoA-I, 1-2 molecules of ApoA-I_(M) dimer or12-40 single helix peptide molecules), 1 molecule of negatively chargedphospholipid and 400 molecules of SM. In other specific embodiments, thenegatively charged lipoprotein complexes are small discoidal disks thatcontain 2-4 ApoA-I equivalents; 1-10, more preferably 3-6, molecule ofnegatively charged phospholipid; and 90-225 molecules, more preferably100-210 molecules, of SM.

6.3.1. Measurement of Complexes and Particle Size

The composition of lipoprotein complexes, as well as their size and thatof lipid particles used in the preparation of the lipoprotein complexes,can be determined using a variety of techniques known in the art.

Protein and lipid concentration of lipoprotein complexes in solution canbe measured by any method known in the art, including, but not limitedto, protein and phospholipid assays, chromatographic methods such asHPLC, gel filtration, GC coupled with various detectors including massspectrometry, UV or diode-array, fluorescent, elastic light scatteringand others. The integrity of lipid and proteins can be also determinedby the same chromatographic techniques as well as by peptide mapping,SDS-page gel electrophoresis, N- and C-terminal sequencing of ApoA-I,and standard assays for determining lipid oxidation.

The lipoprotein complex as well as lipid particles used in thepreparation of the lipoprotein complexes, can range in size as describedherein. Lipid particle size and/or lipid and protein complex size can bedetermined using methods known in the art. Exemplary methods includedynamic light scattering and gel permeation chromatography.

Dynamic light scattering (DLS), also known as photon correlationspectroscopy, measures and the shift in wavelength of a light beamhitting a particle moving in solution by Brownian motion. Specifically,the moving particles scatter light when illuminated by a laser and theresulting intensity fluctuations in the scattered light can be used tocalculate the sphere size distribution in the solution. See, ZetasizerNano Series User Manual, MAN0317 Issue 2.1 (July 2004). DLS determinesthe intensity distribution and average of the particles in solution,based on which particle volume and number distribution and average canbe calculated. The DLS technique can be used to determine the size oflipid particles used to make lipoprotein complexes, as well as the sizeof the lipoprotein complexes themselves. A suitable DLS instrument isthe Zetasizer Nano by Malvern Instruments.

Gel permeation chromatography (GPC) can also be used to determine thesize of protein-containing complexes. Gel permeation chromatographyseparates components in a mixture based on molecular size. The size of alipid-protein complex can be determined by comparing the elution profileof the complex to that of known standards or reference samples,typically by comparison to a calibration curve. Reference samples areavailable commercially and can include both protein and non-proteinstandards, such as albumin, ferritin, and vitamin B₁₂. Current Protocolsin Molecular Biology (1998), Section IV, 10.9.1-10.9.2.

Lipid particles useful in the preparation of the lipoprotein complexesof the disclosure can be at least 45 nm, at least 50 nm, at least 55 nm,at least 60 nm in size, as measured by DLS (e.g., using intensity basedmeasured). Furthermore, lipid particle can be up to 65 nm, up to 70 nm,up to 80 nm, up to 90 nm, up to 100 nm, up to 120 nm, up to 150 nm, upto 200 nm, up to 250 nm, up to 300 nm, or up to 500 nm in size, asmeasured by DLS.

The lipoprotein complexes of the disclosure can range in size from 4 nmto 15 nm, 6 nm to 15 nm, 4 nm to 12 nm, 5 to 12 nm, 6 nm to 12 nm, 8 nmto 12 nm, or 8 nm to 10 nm as measured by the techniques describedherein.

6.4. Populations of Lipoprotein Complexes

The present disclosure further provides populations of the lipoproteincomplexes described herein. The populations comprise a plurality oflipoprotein complexes as described herein, each comprising a proteinfraction and a lipid fraction, e.g. as described above in Section 6.3.Applicants have discovered several features that are thought tocontribute individually or in combination to the potency and the safetyprofile of populations of lipoprotein complexes. Populations oflipoprotein complexes can incorporate any number of the features,described herein alone or in combination.

First, the homogeneity of the lipoprotein complexes in a population,i.e., the prevalence of one or more discrete lipoprotein complex(es) inthe population, as indicated by the one or more discrete peaks oflipoprotein complexes in a population, and the prevalence in lipoproteincomplexes of mature, unmodified apolipoprotein, are thought to increasepotency. Accordingly, the population of lipoprotein complexes cancomprise a protein fraction comprising or consisting essentially of anapolipoprotein, e.g., ApoA-I, and a lipid fraction, where the populationis at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% homogeneous as measured bythe percent of the population in a single peak in gel permeationchromatography.

In some embodiments, lipoprotein complex size in a population can rangebetween 4 nm and 15 nm, e.g., between 5 nm and 12 nm, between 6 nm and15 nm, or between 8 nm and 10 nm.

The apolipoprotein, e.g., ApoA-I, in the population can be mature,preferably full length (untruncated) ApoA-I, and the population cancontain at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% by weightmature, preferably full length (untruncated) ApoA-I. In someembodiments, the population includes no more than 25%, no more than 20%,no more than 15%, no more than 10%, or no more than 5% by weight ofimmature or incompletely processed ApoA-I and/or no more than 20%, nomore than 15%, no more than 10%, or no more than 5% by weight oftruncated ApoA-I.

Second, the purity of apolipoprotein and lipids in the complexes and therelative absence of contaminants in the lipoprotein complexes, alsoreferred to as the purity of the lipoprotein complexes, is thought toreduce the risk of side effects such as liver damage, reflected byincreases in liver enzymes (e.g., transaminase). The purity ofapolipoprotein can be measured by the relative lack of oxidation and/ordeamidation. Accordingly, in certain embodiments, populations oflipoprotein complexes can have reduced amounts of oxidizedapolipoprotein, such as no more than 20%, no more than 15%, no more than10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%,no more than 1% oxidized methionine, especially methionine-112 ormethionine-148, or no more than 15%, no more than 10%, no more than 5%,no more than 4%, no more than 3%, no more than 2%, or no more than 1%oxidized tryptophan. Populations of lipoprotein complexes can also havea reduced percentage of deamidated amino acids, for example no more than15%, no more than 10%, no more than 5%, no more than 4%, no more than3%, no more than 2%, or no more than 1% deaminated amino acids.

It is also desirable to control the purity of the lipids in thelipoprotein complex. Accordingly, in some embodiments, no more than 5%,no more than 4%, no more than 3%, no more than 2% or no more than 1% ofthe lipid in said population is oxidized.

Another measure of the purity of the complexes and populations thereofis a reduction in, or absence of, contaminants that result from themethods of producing or purifying the apolipoprotein or the methods ofmaking the lipoprotein complexes themselves. Accordingly, where theapolipoprotein is purified from host cells, for example mammalian hostcells, the populations of lipoprotein complexes are preferably free ofhost cell DNA or proteins. In specific embodiments, the populationcontains no more than 500 nanograms, no more than 200 nanograms, no morethan 100 nanograms, no more than 50 nanograms, or no more than 20nanograms host cell protein per milligram of the lipoprotein, and/or nomore than 100 picograms, no more than 50 picograms, no more than 25picograms, no more than 10 picograms or no more than 5 picograms hostcell DNA per milligram of the lipoprotein, typically ApoA-I.

Other contaminants that can occur and are to be avoided are endotoxin,which can be present, inter alia, in cell cultures and in plasmasamples, and solvents and detergents, which can be present depending onthe process used to make and/or purify the lipoprotein complex.Populations of lipoprotein complexes can contain at most about 1 EU,about 0.5 EU, about 0.3 EU, or about 0.1 EU of endotoxin per milligramlipoprotein, e.g. ApoA-I. Populations of lipoprotein complexes can alsobe limited to containing no more than 200 ppm, 250 ppm, 100 ppm or anon-aqueous solvent. In a specific embodiment, the population does notcontain any detergent, e.g., cholate.

Additionally, using the methods disclosed herein, it is possible toincorporate most of the apolipoprotein starting material into complexes,limiting the amount of uncomplexed apolipoprotein present in apopulation. The reduction in the amount of uncomplexes apolipoprotein isbeneficial in that it reduces the risk of an immunogenic response due toexposure to a heterologous protein. The population of lipoproteincomplexes can be in a composition in which at least 80%, at least 85%,at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or100% of the protein is in the lipoprotein complex, i.e., the complexedform. Optionally, the population of lipoprotein complexes can be in acomposition in which at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99% or 100% of the lipid is inthe lipoprotein complex.

Optionally, the population can comprise complexes in which the lipidfraction comprises no more than 15%, no more than 10%, no more than 5%,no more than 4%, no more than 3%, no more than 2%, no more than 1%, or0% cholesterol by weight of lipid.

Certain lipid and protein components can form a plurality of differentbut homogeneous lipoprotein complexes. Accordingly, the presentdisclosure also provides compositions comprising two, three, or fourpopulations of lipoprotein complexes comprising different amounts ofapolipoprotein molecules (e.g., two, three or four ApoA-I molecules orApoA-I equivalents). In an exemplary embodiment, a composition comprisestwo lipoprotein complex populations, a first population comprisinglipoprotein complexes having 2 ApoA-I molecules or ApoA-I equivalentsper lipoprotein complex, a second population comprising lipoproteincomplexes having 3 or 4 ApoA-I molecules or ApoA-I equivalents perlipoprotein complex and optionally a third population comprisinglipoprotein complexes having 4 or 3 ApoA-I molecules or ApoA-Iequivalents per lipoproprotein complex, respectively.

The compositions comprising two or more populations of lipoproteincomplexes preferably have low levels of uncomplexed lipoprotein and/orlipid. Accordingly, preferably no more than 15%, no more than 12%, than10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%,no more than 5%, no more than 4%, no more than 3%, no more than 2%, orno more than 1% of the lipid in the composition is in uncomplexed formand/or no more than 15%, no more than 12%, no more than 10%, no morethan 9%, no more than 8%, no more than 7%, no more than 6%, no more than5%, no more than 4%, no more than 3%, no more than 2%, or no more than1% of the lipoprotein in the composition is in uncomplexed form.

Also provided herein are large-scale preparations of lipoproteincomplexes, or populations thereof, that are particularly useful forcommercial applications, such as large scale manufacturing oflipoprotein complexes for therapeutic purposes. The preparationscontemplated herein comprise a population of lipoprotein complexes,e.g., negatively charged lipoprotein complexes, as described herein.

The preparations are provided in volumes, amounts and resultingconcentration of lipoprotein complexes suitable for the manufacturing ofcompositions, e.g., pharmaceutical compositions and dosage forms, on acommercial scale. Typical preparation volumes range from about 5 L toabout 50 L, or more, for example, about 10 L to about 40 L, about 15 Lto about 35 L, about 15 L to about 30 L, about 20 L to about 40 L, about20 L to about 30 L, about 25 L to about 45 L, about 25 L to about 35 L.Preparations can have a volumne of about 5 L, about 6 L, about 7 L,about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L,about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L,about 20 L, about 25 L, about 30 L, about 35 L, about 40 L, about 45 L,or about 50 L. In a preferred embodiment, the preparation has a volumeof about 20 L.

Preparations further contain lipoprotein complexes, or a populationthereof, in amounts sufficient to achieve a concentration ofapolipoprotein ranging from about 5 mg/mL up to about 15 mg/mL, from 5mg/mL to about 10 mg/mL, from about 10 mg/mL to about 15 mg/mL, or about8 mg/mL to about 12 mg/mL of apolipoprotein. Depending on the volume ofthe preparation, amounts can range from about 25 g up to about 350 g,expressed as the amount of apolipoprotein, e.g., ApoA-I, in thepreparation. In a specific embodiment, the preparation contains about 8mg/mL of ApoA-I.

In a specific embodiment, the preparation has a volume of 15 L to 25 Land contains about 100 g to about 250 g of ApoA-I. In another specificembodiment, the preparation has a volume of 30 L to 50 L and containsabout 240 g to about 780 g of ApoA-I.

6.5. Methods of Making Lipoprotein Complexes 6.5.1. Thermal CyclingBased Methods of Making Lipoprotein Complexes

It has been discovered that methods using thermal cycling of protein andlipid components as described herein can be used to generate lipoproteincomplexes with advantages over other methodologies. In the thermalcycling methods provided herein, a protein component and a lipidcomponent, that are subjected to thermal cycling until the majority ofthe protein component (e.g., at least 60%, at least 70%, at least 80%,or at least 90%) is complexed with the lipid component, forming alipoprotein complex. As will be appreciated by skilled artisans, theadvantages of the present methods over other methods for production oflipoprotein complexes include a high complexing efficiency resulting ina substantially homogeneous and pure end product, without few to nobyproducts (e.g., uncomplexed protein) or manufacturing impurities(e.g., detergents or surfactants, degraded proteins, oxidizedcomponents) present in the resulting product, circumventing the need forcostly and wasteful purification steps. Thus, the methods are efficientand result in little-to-no wastage of starting materials. Furthermore,the processes are easy to scale up and have a low equipment cost. Theability to conduct these processes without industrial solvents makesthem environmentally friendly also.

Preferably, to minimize oxidation of the protein and lipid components,one, more than one or all steps of complex formation (includinghomogenization of the lipid component) are carried out under an inertgas (e.g., nitrogen, argon or helium) blanket.

6.5.2. The Lipid Component

The thermal cycling methods of the disclosure can utilize a variety oflipids, alone or in combination, including saturated, unsaturated,natural and synthetic lipids and/or phospholipids, as described above inSection 6.2.

The lipids can be prepared for thermal cycling with the proteincomponent using any method that generates lipid particles, such asmultilamellar vesicles (“MLVs”), small unilamellar vesicles (“SUVs”),large unilamellar vesicles (“LUVs”), micelles, dispersions and the like.

A range of technologies is known for producing lipid particles. Lipidparticles have been produced using a variety of protocols, formingdifferent types of vesicles. It is preferred that the particles used inthe thermal cycling methods of the disclosure are predominantly in the45-80 nm size range, most preferably in the 55 nm to 75 nm size range.

High pressure homogenization, for example microfluidization,advantageously produces particles of suitable sizes. The homogenizationpressure is preferably at least 1,000 bars, at least 1,200 bars, atleast 1,400 bars, at least 1,600 bars, at least 1,800 bars, and is mostpreferably at least 2,000 bars, for example at least 2,200 bars, atleast 2,400 bars, at least 2,600 bars, at least 2,800 bars, at least3,000 bars, at least 3,200 bars, at least 3,400 bars, at least 3,600bars, at least 3,800 bars, or at least 4,000 bars. In specificembodiments, the homogenization pressure is in a range between any pairof the foregoing values, e.g., 1,600 to 3,200 bars; 1,800 to 2,800 bars;1,900 to 2,500 bars; 2,000 to 2,500 bars; 2,000 to 3,000 bars; 2,400 to3,800 bars; 2,800 to 3,400 bars; and so on and so forth. One bar equals100 kPa, 1,000,000 dynes per square centimeter (baryes), 0.987 atm(atmospheres), 14.5038 psi, 29.53 inHg and 750.06 torr.

In one suitable homogenization method, an emulsion of the lipids istransferred into the feed vessel of a Microfluidizer Model 110Y(Microfluidics Inc, Newton, Mass.). The unit is immersed in a bath tomaintain the process temperature (e.g., 55° C., 58° C., 62° C., etc.)during homogenization, and is flushed with an inert gas such as argonbefore use. After priming, the emulsion is passed through thehomogenizer in continuous re-cycle for 5-20 minutes at a pressuregradient across the interaction head. Homogenization of the lipidcomponent in the absence of the protein component avoids the destructionof the protein component by high shear used in homogenizationtechniques.

Other methods can suitably be used, provided that particles of asuitable size can be obtained. For example, hydration of lipids byaqueous solution can result in the dispersion of lipids and spontaneousformation of multimellar vesicles (“MLVs”). An MLV is a particle withmultiple lipid bilayers surrounding the central aqueous core. Thesetypes of particles are larger than small unilamellar vesicles (SUVS) andmay be 350-400 nm in diameter. MLVs can be prepared by solubilizinglipids in chloroform in a round-bottom flask and evaporating thechloroform until the lipid formed a thin layer on the wall of the flask.The aqueous solution is added and the lipid layer is allowed torehydrate. Vesicles formed as the flask is swirled or vortexed. Deameret al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York(citing Bangham et al., 1965, J. Mol. Biol. 13:238). This method canalso be used to generate single lamellar vesicles. Johnson et al., 1971,Biochim. Biophys. Acta 233:820.

A small unilamellar vesicle (SUV) is a particle with a single lipidbilayer enclosing an aqueous core. Depending on the method employed togenerate the SUVS, they can range in size from 25-110 nm in diameter.The first SUVs were prepared by drying a phospholipid preparation inchloroform under nitrogen, adding the aqueous layer to produce a lipidconcentration in the millimolar range, and sonicating the solution at45° C. to clarity. Deamer et al., 1983, in Liposomes (Ostro, Ed.),Marcel Dekker, Inc. New York. SUVs prepared in this fashion yieldparticles in the range of 25-50 nm in diameter.

Another method of making SUVs is rapidly injecting an ethanol/lipidsolution into the aqueous solution to be encapsulated. Deamer et al.,1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citingBatzri et al., 1973, Biochim. Biophys. Acta 298:1015). SUVs produced bythis method range in size from 30-110 nm in diameter.

SUVs can also be produced by passing multilamellar vesicles through aFrench Press four times at 20,000 psi. The SUVs produced will range insize from 30-50 rim in diameter. Deamer et al., 1983, in Liposomes(Ostro, Ed.), Marcel Dekker, Inc. New York (citing Barenholz et al.,1979, FEBS Letters 99:210).

Multilamellar and unilamellar phospholipid vesicles can also be formedby extrusion of aqueous preparations of phospholipids at high pressurethrough small-pore membranes (Hope et al., 1996, Chemistry and Physicsof Lipids, 40:89-107)

Large unilamellar vesicles are similar to SUVs in that they are singlelipid bilayers surrounding the central aqueous core, but LUVs are muchlarger that SUVs. Depending on their constituent parts and the methodused to prepare them, LUVs can range in size from 50-1000 nm indiameter. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker,Inc. New York. LUVs are usually prepared using one of three methods:detergent dilution, reverse-phase evaporation, and infusion.

In the detergent dilution technique, detergent solutions such ascholate, deoxycholate, octyl glucoside, heptyl glucoside and TritonX-100 are used to form micelles from the lipid preparation. The solutionis then dialyzed to remove the detergent. Deamer et al., 1983, inLiposomes (Ostro, Ed.), Marcel Dekker, Inc. New York.

The reverse-phase evaporation technique solubilizes lipid inaqueous-nonpolar solutions, forming inverted micelles. The nonpolarsolvent is evaporated and the micelles aggregate to form LUVs. Thismethod generally requires a great deal of lipid.

The infusion method injects a lipid solubilized in a non-polar solutioninto the aqueous solution to be encapsulated. As the nonpolar solutionevaporates, lipids collect on the gas/aqueous interface. The lipidsheets form LUVs and oligolamellar particles as the gas bubbles throughthe aqueous solution. Particles are sized by filtration. Deamer et al.,1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citingDeamer et al., 1976, Biochim. Biophys. Acta 443:629 and Schieren et al.,1978, Biochim. Biophys. Acta 542:137).

An aliquot of the resulting lipid preparation can be characterized toconfirm that the lipid particles are suitable for use as the lipidcomponent in the thermocyling methods disclosed herein. Characterizationof the lipid preparation can be performed using any method known in theart, including, but not limited to, size exclusion filtration, gelfiltration, column filtration, gel permeation chromatography, andnon-denaturating gel electrophoresis.

6.5.3. The Protein Component

The protein component of the lipoprotein complexes is not critical forsuccess in the present thermal cycling methods. Virtually anylipid-binding protein, such as an apolipoprotein and/or derivative oranalog thereof that provides therapeutic and/or prophylactic benefit canbe included in the complexes. Moreover, any alpha-helical peptide orpeptide analog, or any other type of molecule that “mimics” the activityof an apolipoprotein (such as, for example ApoA-I) in that it canactivate LCAT or form discoidal particles when associated with lipids,can be included in the lipoprotein complexes, and is therefore includedwithin the definition of “lipid-binding protein.” The lipid-bindingproteins that can be used in the thermal cycling methods include thosein described in Section 6.1 above. The lipid-binding proteins can berecombinantly produced as described in Section 6.1.2 above. Thelipid-binding proteins can be purified by any of the methods describedherein, including as described in Section 6.1.3 or Section 6.1.4 above.

The protein component can be purified from animal sources (and inparticular from human sources), chemically synthesized or producedrecombinantly as is well-known in the art, see, e.g., Chung et al.,1980, J. Lipid Res. 21(3):284-91; Cheung et al., 1987, J. Lipid Res.28(8):913-29. See also U.S. Pat. Nos. 5,059,528, 5,128,318, 6,617,134;U.S. Publication Nos. 20002/0156007, 2004/0067873, 2004/0077541, and2004/0266660; and PCT Publications Nos. WO/2008/104890 andWO/2007/023476.

The protein component can include lipids in protein/peptide to lipidratio that is at least 5-fold greater (e.g., at least 5-fold, at least10-fold or at least 20-fold greater) than the protein/peptide to lipidratio in the desired complex. For example, to produce a lipoproteincomplex in which the desired protein to lipid ratio is 1:200 on a molarbasis, the protein in the protein component can be combined with alipid, typically one that will represent only a small fraction of thelipid in the final complex, e.g., in a ratio of 1:10 to 1:20. Withoutimplying any mechanism, this “pre-” complexing of the protein to a smallamount of lipid is useful when the desired complex has more than onetype of lipid, allowing more homogeneous distribution of a lipid that ispresent in small quantities in the lipoprotein complex produced bythermal cycling (e.g., 10% or less by weight of total lipid, 5% or lessby weight of total lipid, 3% or less by weight of total lipid, 2% orless by weight of total lipid, or 1% or less by weight of total lipid inthe desired lipoprotein complex).

6.5.4. Generating Lipoprotein Complexes by Thermal Cycling

The methods generally entail thermally cycling a suspension comprisinglipid particles and lipid binding proteins between a “high” temperaturerange and a “low” temperature range until lipoprotein complexes areformed.

The suspension that is thermally cycled is contains a lipid componentand a protein component that are brought together, preferably at atemperature in the high temperature range, to form a “starting”suspension that is then subject to thermal cycling.

The optimum ratio of lipids and proteins in the starting suspension isdetermined by the desired stoichiometry of the components in theultimate lipoprotein complexes to be produced. As will be recognized byskilled artisans, the molar ratio of the lipid fraction to the proteinfraction will depend upon, among other factors, the identity(ies) of theproteins and/or peptides in the protein component, the identities andquantities of the lipids in the lipid fraction, and the desired size ofthe lipoprotein complex. Suitable lipid to protein ratios in thelipoprotein complexes can be determined using any number of functionalassays known in the art, including, but not limited to, gelelectrophoresis mobility assay, size exclusion chromatography,interaction with HDL receptors, recognition by ATP-binding cassettetransporter (ABCAl), uptake by the liver, andpharmacokinetics/pharmacodynamics. For example, gel electrophoresismobility assays can be used to determine the optimum ratio of lipidcomponent to protein component in the complexes. Where the complexesproduced by the methods of the disclosure are charged, as a result ofinclusion of phospholipids in the lipid component, the complexes can bedesigned to exhibit an electrophoretic mobility that is similar tonatural pre-beta-HDL or alpha-HDL particles. Thus, in some embodiments,natural pre-beta-HDL or alpha-HDL particles can be used as standard fordetermining the mobility of the complexes.

In a preferred embodiment, the ultimate complex has at least oneapolipoprotein or apoliprotein mimic (most preferably mature humanApoA-I or ApoA-I peptide, respectively), at least one neutral lipid, andat least one negatively charged lipid, such as those described in PCTWO2006/100567, the contents of which are incorporated by referenceherein.

Because the biological activity of apolipoproteins such as ApoA-I arethought to be mediated by the amphipathic helices comprising theapolipoprotein, it is convenient to express the apolipoprotein fractionof the lipid:apolipoprotein molar ratio using ApoA-I proteinequivalents. It is generally accepted that ApoA-I contains 6-10amphipathic helices, depending upon the method used to calculate thehelices. Other apolipoproteins can be expressed in terms of ApoA-Iequivalents based upon the number of amphipathic helices they contain.For example, ApoA-I_(M), which typically exists as a disulfide bridgeddimer, can be expressed as 2 ApoA-I equivalents, because each moleculeof ApoA-I_(M) contains twice as many amphipathic helices as a moleculeof ApoA-I. Conversely, a peptide apolipoprotein that contains a singleamphipathic helix can be expressed as a 1/10 to 1/6 ApoA-I equivalent,because each molecule contains 1/10 to 1/6 as many amphipathic helicesas a molecule of ApoA-I.

In general, the lipid:ApoA-I equivalent molar ratio of the lipoproteincomplexes (defined herein as “Ri”) will range from about 2:1 to 200:1.In some embodiments, the Ri is about from 50:1 to 150:1, or from 75:1 to125:1, from 10:1 to 175:1. Ratios in weight can be obtained using a MWof approximately 650-800 for phospholipids.

In certain embodiments, the molar ratio of the components is 2-6(negatively charged lipid, e.g., DPPG): 90-120 (neutral lipid, e.g.,SM): 1 (ApoA-I equivalents). In a specific embodiment, described inExample 1, the complex comprises DPPG, SM and ApoA-I in a lipid toprotein molar ratio of approximately 108:1, with DPPG representing 3%(+/−1%) of the total lipid by weight and SM representing 97% (+/−5%) ofthe lipid by weight.

The concentration of the lipid and protein components in the startingsuspension prior to the initiation of thermocycling can range from 1 to30 mg/ml concentration of ApoA-I equivalents and from 1 to 100 mg/mlconcentrations of lipid. In specific embodiments, the concentration ofthe protein component is selected from 1 to 30 mg/ml, 2 to 20 mg/ml,from 5 to 20 mg/ml, from 2 to 10 mg/ml, from 5 to 15 mg/ml, from 5 to 20mg/ml, and from 10 to 20 mg/ml, and the concentration of the lipidcomponent is independently selected from 10 to 100 mg/ml, from 10 to 75mg/ml, from 25 to 50 mg/ml, from 10 to 75 mg/ml, from 25 to 100 mg/ml,from 25 to 75 mg/ml, and from 1 to 75 mg/ml.

The high and low temperature ranges of the thermocycling process arebased on the phase transition temperatures of the lipid and proteincomponents of the lipoprotein complexes. Alternatively, where the lipidcomponent does not exhibit a defined or discrete phase transition, ascould occur when using phospholipids having unsaturated fatty acidchains or a mixture of phospholipids, the high and low temperatureranges of the thermocycling differ by at least about 20° C., up to about40° C. or even more. For example, in some embodiments, the low and hightemperature ranges differ by 20° C.-30° C., 20° C.-40° C., 20° C.-50°C., 30° C.-40° C., 30° C.-50° C., 25° C.-45° C., 35° C.-55° C.

For a lipid, the phase transition involves a change from a closelypacked, ordered structure, known as the gel state, to a loosely packed,less-ordered structure, known as the fluid state. Lipoprotein complexesare typically formed in the art by incubating lipid particles andapolipoproteins at temperatures near the transition temperature of theparticular lipid or mixture of lipids used. The phase transitiontemperature of the lipid component (which can be determined bycalorimetry) +/−5° C.-10° C. represents the “low” temperature range inthe methods of the disclosure.

For a protein, the phase transition temperature involves a change fromthe folded three dimensional structure into a two-dimensional structure.For a lipid, the phase transition involves a change from a closelypacked, ordered structure, known as the gel state, to a loosely packed,less-ordered structure, known as the fluid state. Lipoprotein complexesare typically formed in the art by incubating lipid particles andapolipoproteins at temperatures near the transition temperature of theparticular lipid or mixture of lipids used.

The phase transition temperature of the lipid component (which can bedetermined by calorimetry) +/−12° C., more preferably +/−10° C.,represents the “low” temperature range in the methods of the disclosure.In certain embodiments, the low temperature range is +/−3° C., +/−5° C.,or +/−8° C. of the phase transition temperature of the lipid component.In one specific embodiment, the low temperature range is from no lessthan 5° C. or no less than 10° C. below to 5° C. above the phasetransition temperature of the lipid component.

For a protein, the phase transition temperature involves a change fromthe tertiary structure into the secondary structure. The phasetransition temperature of the protein component +/−12° C., morepreferably +/−10° C., represents the “high” temperature range in themethods of the disclosure. In specific embodiments, the high temperaturerange is +/−3° C., +/−5° C., or +/−8° C. of the phase transitiontemperature of the protein component. In one specific embodiment, thelow temperature range is from 10° C. below to no more than 5° C., nomore than 10° C., or no more than 15° C. above the phase transitiontemperature of the protein component.

The starting suspension is subjected to thermal cycling between the hightemperature and the low temperature, preferably starting at the hightemperature, until at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% of theprotein in the starting suspension is incorporated into lipoproteincomplexes. Using suitable stoichiometric quantities of lipid and proteincomponents, substantially complete complexation of the lipid and proteincomponents can be reached after several cycles. The number of cycleswill depend on the protein and lipid components, the duration of thecycles, but typically 5 or more cycles, 10 or more cycles, or 15 or morecycles (at both the high and low temperatures) will be required forsubstantially complete complexation. The cycles typically range from 2minutes to 60 minutes. In specific embodiments, the cycles range from 5to 30 minutes, from 10 to 20 minutes, from 5 to 20 minutes, from 2 to 45minutes, or from 5 to 45 minutes at each temperature.

The complexes produced by the methods are typically supramolecularassemblies shaped as micelles, vesicles, spherical or discoidalparticles in which the protein component is physically bound tophospholipids at a specific stoichiometric range between thephospholipid and protein and with a homogeneous size distribution. Thepresent methods advantageously result in substantially completecomplexation of the lipids and/or proteins in the starting suspension,resulting in a composition that is substantially free lipids and/or freeprotein, as observed by separation methods such as chromatography. Thus,the methods of the disclosure can be performed in the absence of apurification step.

The methods of the disclosure advantageously produce complexes that arehomogenous in their size distribution, circumventing the need for sizefractionation.

In some embodiments of the disclosure, the lipoprotein complexes willcontain more than one type of lipid, including one or more lipids inrelatively small quantities (e.g., less than 10%, less than 5%, lessthan 3% or less than 1% of the lipid component). To optimize dispersion,lipids used in small quantities can be pre-blended with the proteincomponent rather than incorporated into the lipid particles in the lipidcomponent.

An aliquot of the resulting lipoprotein complexes can be characterizedto confirm that the complexes possess the desired characteristics, e.g.,substantially complete (e.g., >90%, >95%, >97% or >98%) incorporation ofthe protein component into the lipid component. Characterization of thecomplexes can be performed using any method known in the art, including,but not limited to, size exclusion filtration, gel filtration, columnfiltration, gel permeation chromatography, and non-denaturating gelelectrophoresis.

The homogeneity and/or stability of the lipoprotein complexes orcomposition described herein can be measured by any method known in theart, including, but not limited to, chromatographic methods such as gelfiltration chromatography. For example, in some embodiments a singlepeak or a limited number of peaks can be associated with a stablecomplex. The stability of the complexes can be determined by monitoringthe appearance of new of peaks over time. The appearance of new peaks isa sign of reorganization among the complexes due to the instability ofthe particles.

Preferably, to minimize oxidation of the protein and lipid components,the thermocycling is carried out under an inert gas (e.g., nitrogen,argon or helium) blanket.

6.5.5. Other Methods of Making Lipoprotein Complexes

The lipoprotein complexes described herein, including negatively chargedlipoprotein complexes, can be prepared in a variety of forms, including,but not limited to vesicles, liposomes, proteoliposomes, micelles, anddiscoidal particles. In addition to the thermal cycling methodsdescribed above, a variety of methods known to those skilled in the artcan be used to prepare the lipoprotein complexes. Various techniques forpreparing liposomes or proteoliposomes may be used. For example,apolipoprotein can be co-sonicated (using an ultrasonic bath orultrasonic probe) with the appropriate phospholipids to form complexes.Alternatively, apolipoprotein, e.g., ApoA-I, can be combined withpreformed lipid vesicles resulting in the spontaneous formation oflipoprotein complexes. The lipoprotein complexes can also be formed by adetergent dialysis method; e.g., a mixture of apolipoprotein, chargedphospholipid(s), and SM and a detergent such as cholate is dialyzed toremove the detergent and reconstituted to form negatively chargedlipoprotein complexes (see, e.g., Jonas et al., 1986, Methods inEnzymol. 128:553-82), or by using an extruder device or byhomogenization.

In some embodiments, complexes are prepared by homogenization using highpressure (e.g., about 32000 p.s.i.) for about 40, about 50, or about 60minutes. In a specific embodiment, a complex comprising ApoA-I, SN, andDPPG is prepared as follows. ApoA-I is dissolved in phosphate buffer andincubated at 50° C. with a dispersion of DPPG in phosphate buffer madeusing a high shear mixer. The ApoA-I/DPPG mixture is then combined witha dispersion of SM, and homogenized at pressures over 30,000 p.s.i. at30-50° C. until complex formation is substantially complete, asmonitored by dynamic light scattering or gel permeation chromatography.

In some embodiments, lipoprotein complexes can be prepared by thecholate dispersion method described in Example 1 of U.S. publication2004/0067873, the disclosure of which is incorporated herein byreference. Briefly, dry lipid is hydrated in NaHCO₃ buffer, thenvortexed and sonicated until all lipid is dispersed. Cholate solution isadded, the mixture is incubated for 30 minutes, with periodic vortexingand sonicating, until it turns clear, indicating that the lipid cholatemicelles are formed. ProApoA-I in NaHCO₃ buffer is added, and thesolution incubated for 1 hour at approximately 37° C.-50° C.

Cholate can be removed by methods well known in the art. For examplecholate can be removed by dialysis, ultrafiltration or by removal ofcholate molecules by adsorption absorption onto an affinity bead orresin. In one embodiment, the affinity beads, e.g., BIO-BEADS® (Bio-RadLaboratories) are added to the preparation of negatively chargedlipoprotein complexes and cholate to adsorb the cholate. In anotherembodiment, the preparation, e.g., a micellar preparation of thelipoprotein complexes and cholate, is passed over a column packed withaffinity beads.

In a specific embodiment, cholate is removed from a preparation oflipoprotein complexes by loading the preparation onto BIO-BEADS® withina syringe. The syringe is then sealed with barrier film and incubatedwith rocking at 4° C. overnight. Before use, the cholate is removed byinjecting the solution through BIO-BEADS®, where it is adsorbed by thebeads.

The lipoprotein complexes, such as negatively charged lipoproteincomplexes described herein, are expected to have an increased half-lifein the circulation when the complexes have a similar size and density toHDL, especially to the HDLs in the pre-beta-1 or pre-beta-2 HDLpopulations. Stable preparations having a long shelf life may be made bylyophilization. In some embodiments, co-lyophilization methods commonlyknown in the art are used to prepare ApoA-I-lipid complexes. Briefly,the co-lyophilization steps include either solubilizing a mixture ofApoA-I and lipid in organic solvent or solvent mixture, or solubilizingApoA-I and lipid separately and mixing them together. Desirablecharacteristics of solvents or solvent mixtures for co-lyophilizationinclude: (i) a medium relative polarity to be able to dissolvehydrophobic lipids and amphipathic protein, (ii) class 2 or class 3solvents according to FDA solvent guidelines (Federal Register, volume62, No. 247) to avoid potential toxicity associated with residualorganic solvent, (iii) low boiling point to assure ease of solventremoval during lyophilization, (iv) high melting point to provide forfaster freezing, higher temperatures of condensation and less wear andtear on the freeze-dryer. In some embodiments, glacial acetic acid isused. Combinations of methanol, glacial acetic acid, xylene, orcyclohexane can also be used.

The ApoA-I-lipid solution is then lyophilized to obtain a homogeneouspowder. The lyophilization conditions can be optimized to obtain fastevaporation of solvent with minimal amount of residual solvent in thelyophilized apolipoprotein-lipid powder. The selection of freeze-dryingconditions can be determined by the skilled artisan, depending on thenature of solvent, type and dimensions of the receptacle, holdingsolution, fill volume, and characteristics of the freeze-dryer used.

The lyophilized lipoprotein complexes can be used to prepare bulksupplies for pharmaceutical reformulation, or to prepare individualaliquots or dosage units that can be reconstituted to obtain a solutionor suspension of lipoprotein complexes. For reconstitution, thelyophilized powder is rehydrated with an aqueous solution to a suitablevolume (e.g., 5 mg polypeptide/ml, which is convenient for intravenousinjection). In some embodiments, the lyophilized powder is rehydratedwith phosphate buffered saline or a physiological saline solution. Themixture can be agitated or vortexed to facilitate rehydration. Thereconstitution step can be performed at a temperature equal to orgreater than the phase transition temperature of the lipid component ofthe complexes.

The ApoA-I-lipid complexes can form spontaneously after hydration oflyophilized apolipoprotein-lipid powder with an aqueous medium ofappropriate pH and osmolality. In some embodiments, the hydration mediumcontains stabilizers selected from, but not limited to, sucrose,trehalose and glycerin. In some embodiments, the solution is heatedseveral times above the transition temperature of the lipids in orderfor complexes to form. The ratio of lipid to protein can be from 1:1 to200:1 (mole/mole), and is preferably 3:1 to 2:1 lipid:protein (w/w),more preferably 2.7:1 to 2.1:1 lipid:protein (w/w), e.g., 2.7:1lipid:protein (w/w). The powder is hydrated to obtain a final complexconcentration of 5-30 mg/ml expressed in protein equivalents.

In various embodiments, ApoA-I powder can be obtained by freeze-dryingthe polypeptide solution in NH₄HCO₃ aqueous solution. A homogeneoussolution of ApoA-I and lipid (e.g., sphingomyelin) is then formed bydissolving the lipid powder and the ApoA-I powder in glacial aceticacid. The solution is then lyophilized, and HDL-likeapolipoprotein-lipid complexes are formed by hydration of the resultingpowder in an aqueous medium.

In some embodiments, ApoA-I-lipid complexes are formed byco-lyophilization of phospholipid and protein solutions or suspensions.A homogeneous solution of ApoA-I and lipid (e.g., phospholipids) in anorganic solvent or organic solvent mixture is lyophilized, andApoA-I-lipid complexes are subsequently formed spontaneously byhydration of the lyophilized powder in an aqueous buffer. Examples oforganic solvents and solvent mixtures for use in this method include,but are not limited to, acetic acid, an acetic acid/xylene mixture, anacetic acid/cyclohexane mixture, and a methanol/xylene mixture.

An aliquot of the resulting reconstituted preparations can becharacterized to confirm that the complexes have the desired sizedistribution; e.g., the size distribution of HDL. An exemplary methodfor characterizing the size is gel filtration chromatography. A seriesof proteins of known molecular weight and Stokes' diameter, as well ashuman HDL, can be used as standards to calibrate the column.

In other embodiments, recombinant ApoA-I-lipid complexes are made bycomplexing ApoA-I with the lipids disclosed in U.S. patent publicationno. 2006/0217312 and international publication no. WO 2006/100567(PCT/IB2006/000635), the disclosures of which are incorporated herein byreference.

U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166 and 6,287,590(incorporated herein by reference in their entireties) disclose a simplemethod for preparing negatively charged lipoprotein complexes that havecharacteristics similar to HDL. This method, which involvesco-lyophilization of apolipoprotein and lipid solutions in organicsolvent (or solvent mixtures) and formation of negatively chargedlipoprotein complexes during hydration of the lyophilized powder, hasthe following advantages: (1) the method requires very few steps; (2)the method uses inexpensive solvent(s); (3) most or all of the includedingredients are used to form the designed complexes, thus avoiding wasteof starting material that is common to the other methods; (4)lyophilized complexes are formed that are very stable during storagesuch that the resulting complexes may be reconstituted immediatelybefore use; (5) the resulting complexes usually need not be furtherpurified after formation and before use; (6) toxic compounds, includingdetergents such as cholate, are avoided; and (7) the production methodcan be easily scaled up and is suitable for GMP manufacture (i.e., in anendotoxin-free environment).

Other suitable methods are described in US published application no.2006/0217312 and international publication WO 2006/100567 (PCT/IB2006/000635), the disclosure of each of which is incorporated herein byreference.

Preferably, to minimize oxidation of the protein and lipid components,one, more than one or all steps of complex formation are carried outunder an inert gas (e.g., nitrogen, argon or helium) blanket.

Protein and lipid concentration of apolipoprotein-lipid particles insolution can be measured by any method known in the art, including, butnot limited to, protein and phospholipid assays, chromatographic methodssuch as HPLC, gel filtration, GC coupled with various detectorsincluding mass spectrometry, UV or diode-array, fluorescent, elasticlight scattering and others. The integrity of lipid and proteins can bealso determined by the same chromatographic techniques as well as bypeptide mapping, SDS-page gel electrophoresis, N- and C-terminalsequencing of ApoA-I, and standard assays for determining lipidoxidation.

6.6. Pharmaceutical Compositions

The pharmaceutical compositions contemplated by the disclosure comprisenegatively charged lipoprotein complexes as the active ingredient in apharmaceutically acceptable carrier suitable for administration anddelivery in vivo. Since peptides may comprise acidic and/or basictermini and/or side chains, peptide mimetic apolipoproteins can beincluded in the compositions in either the form of free acids or bases,or in the form of pharmaceutically acceptable salts. Modified proteinssuch as amidated, acylated, acetylated or pegylated proteins, may alsobe used. Optionally, the pharmaceutical compositions can compriselipoprotein complexes loaded with one or more hydrophobic, lipophilic,or apolar active agents, as described above in Sections 6.2 and 6.3.

Injectable compositions include sterile suspensions, solutions oremulsions of the active ingredient in aqueous or oily vehicles. Thecompositions can also comprise formulating agents, such as suspending,stabilizing and/or dispersing agent. In some embodiments, the injectablecomposition comprises negatively charged lipoprotein complexes inphosphate buffered saline (10 mM sodium phosphate, 80 mg/mL sucrose, pH8.2). The compositions for injection can be presented in unit dosageform, e.g., in ampules or in multidose containers, and can compriseadded preservatives. For infusion, a composition can be supplied in aninfusion bag made of material compatible with negatively chargedlipoprotein complexes, such as ethylene vinyl acetate or any othercompatible material known in the art.

Suitable dosage forms comprise negatively charged lipoprotein complexesat a final concentration of about 5 mg/mL to about 15 mg/mL oflipoprotein. In a specific embodiment, the dosage form comprisesnegatively charged lipoprotein complexes at a final concentration ofabout 8 mg/mL to about 10 mg/mL Apolipoprotein A-I, preferably about 8mg/mL.

Preferably, to minimize oxidation of the protein and lipid components,the pharmaceutical compositions are formulated and/or filled under aninert gas (e.g., nitrogen, argon or helium) blanket.

6.7. Methods of Treatment

The lipoprotein complexes, e.g., negatively charged lipoproteincomplexes, and compositions described herein can be used for virtuallyevery purpose lipoprotein complexes have been shown to be useful.Lipoprotein complexes, such as negatively charged lipoprotein complexes,are effective at mobilizing cholesterol, even when administered at dosessignificantly lower than the amounts of apolipoprotein (20 mg/kg to 100mg/kg per administration every 2 to 5 days, 1.4 g to 8 g per averagesized human) required by currently available treatment regimens.

Consequently, the complexes and compositions of the present disclosureare particularly useful to treat or prevent cardiovascular diseases,disorders, and/or associated conditions. Methods of treating orpreventing a cardiovascular disease, disorder, and/or associatedcondition in a subject generally comprise administering to the subject alow (<15 mg/kg) dose or amount of a lipoprotein complex orpharmaceutical composition described herein according to a regimeneffective to treat or prevent the particular indication.

Lipoprotein complexes are administered in an amount sufficient oreffective to provide a therapeutic benefit. In the context of treating acardiovascular disease, disorder, and/or associated condition, atherapeutic benefit can be inferred if one or more of the followingoccurs: an increase in cholesterol mobilization as compared to abaseline, a reduction in atherosclerotic plaque volume, an increase inhigh density lipoprotein (HDL) fraction of free cholesterol as comparedto a baseline level, without an increase in mean plasma triglycerideconcentration or an increase above normal range of liver transaminase(or alanine aminotransferase) levels. A complete cure, while desirable,is not required for therapeutic benefit to exist.

In some embodiments, the lipoprotein complex is administered at a doseof about 2 mg/kg ApoA-I equivalents to about 12 mg/kg ApoA-I equivalentsper injection. In some embodiments, the lipoprotein complex isadministered at a dose of about 3 mg/kg ApoA-I equivalents. In someembodiments, the lipoprotein complex is administered at a dose of about6 mg/kg ApoA-I equivalents. In some embodiments, the lipoprotein complexis administered at a dose of about 12 mg/kg ApoA-I equivalents.

Subjects to be treated are individuals suffering from a preventing acardiovascular disease, disorder, and/or associated condition.Non-limiting examples of such cardiovascular diseases, disorders and/orassociated conditions that can be treated or prevented with thelipoprotein complexes and compositions described herein include,peripheral vascular disease, restenosis, atherosclerosis, and the myriadclinical manifestations of atherosclerosis, such as, for example,stroke, ischemic stroke, transient ischemic attack, myocardialinfarction, acute coronary syndrome, angina pectoris, intermittentclaudication, critical limb ischemia, valve stenosis, and atrial valvesclerosis. Subjects can be individuals with a prior incidence of acutecoronary syndrome, such as a myocardial infarction (either with orwithout ST elevation) or unstable angina. The subject treated may be anyanimal, for example, a mammal, particularly a human.

In one embodiment, the methods encompass a method of treating orpreventing a cardiovascular disease, comprising administering to asubject a charged lipoprotein complex or composition described herein inan amount that does not alter a patient's baseline ApoA-I followingadministration.

In other embodiments, the methods encompass a method of treating orpreventing a cardiovascular disease, comprising administering to asubject a lipoprotein complex (e.g., a charged complex) or compositiondescribed herein in an amount that is effective to achieve a serum levelof free or complexed apolipoprotein higher than a baseline (initial)level prior to administration by about 5 mg/dL to 100 mg/dLapproximately to two hours after administration and/or by about 5 mg/dLto 20 mg/dL approximately 24 hours after administration.

In another embodiment, the methods encompass a method of treating orpreventing a cardiovascular disease, comprising administering to asubject a lipoprotein complex (e.g., a charged complex) or compositiondescribed herein in an amount effective to achieve a circulating plasmaconcentrations of a HDL-cholesterol fraction for at least one dayfollowing administration that is at least about 10% higher than aninitial HDL-cholesterol fraction prior to administration.

In another embodiment, the methods encompass a method of treating orpreventing a cardiovascular disease, comprising administering to asubject a lipoprotein complex (e.g., a charged complex) or compositiondescribed herein in an amount effective to achieve a circulating plasmaconcentration of a HDL-cholesterol fraction that is between 30 and 300mg/dL between 5 minutes and 1 day after administration.

In another embodiment, the methods encompass a method of treating orpreventing a cardiovascular disease, comprising administering to asubject a lipoprotein complex (e.g., a charged complex) or compositiondescribed herein in an amount effective to achieve a circulating plasmaconcentration of cholesteryl esters that is between 30 and 300 mg/dLbetween 5 minutes and 1 day after administration.

In still another embodiment, the methods encompasses a method attreating or protecting a cardiovascular disease, comprisingadministering to a subject a lipoprotein complex (e.g., a chargedcomplex) or composition described herein in an amount effective toachieve an increase in fecal cholesterol excretion for at least one dayfollowing administration that is at least about 10% above a baseline(initial) level prior to administration.

The lipoprotein complexes, including negatively charged lipoproteincomplexes, or compositions described herein can be used alone or incombination therapy with other drugs used to treat or prevent theforegoing conditions. Such therapies include, but are not limited tosimultaneous or sequential administration of the drugs involved. Forexample, in the treatment of hypercholesterolemia, such as familialhypercholesterolemia (homozygous or heterozygous) or atherosclerosis,charged lipoprotein formulations can be administered with any one ormore of the cholesterol lowering therapies currently in use; e.g.,bile-acid resins, niacin, statins, inhibitors of cholesterol absorptionand/or fibrates. Such a combined regimen may produce particularlybeneficial therapeutic effects since each drug acts on a differenttarget in cholesterol synthesis and transport; i.e., bile-acid resinsaffect cholesterol recycling, the chylomicron and LDL population; niacinprimarily affects the VLDL and LDL population; the statins inhibitcholesterol synthesis, decreasing the LDL population (and perhapsincreasing LDL receptor expression); whereas the lipoprotein complexes,including negatively charged lipoprotein complexes, described hereinaffect RCT, increase HDL, and promote cholesterol efflux.

In another embodiment, the lipoprotein complexes, including negativelycharged lipoprotein complexes, or compositions described herein may beused in conjunction with fibrates to treat or prevent coronary heartdisease; coronary artery disease; cardiovascular disease, restenosis,vascular or perivascular diseases; atherosclerosis (including treatmentand prevention of atherosclerosis); Exemplary formulations and treatmentregimens are described below.

The lipoprotein complexes, including negatively charged lipoproteincomplexes, or compositions described herein can be administered indosages that increase the small HDL fraction, for example, the pre-beta,pre-gamma and pre-beta-like HDL fraction, the alpha HDL fraction, theHDL3 and/or the HDL2 fraction. In some embodiments, the dosages areeffective to achieve atherosclerotic plaque reduction as measured by,for example, imaging techniques such as magnetic resonance imaging (MRI)or intravascular ultrasound (IVUS). Parameters to follow by IVUSinclude, but are not limited to, change in percent atheroma volume frombaseline and change in total atheroma volume. Parameters to follow byMRI include, but are not limited to, those for IVUS and lipidcomposition and calcification of the plaque.

The plaque regression could be measured using the patient as its owncontrol (time zero versus time t at the end of the last infusion, orwithin weeks after the last infusion, or within 3 months, 6 months, or 1year after the start of therapy.

Administration can best be achieved by parenteral routes ofadministration, including intravenous (IV), intramuscular (IM),intradermal, subcutaneous (SC), and intraperitoneal (IP) injections. Incertain embodiments, administration is by a perfusor, an infiltrator ora catheter. In some embodiments, the lipoprotein complexes, e.g.,negatively charged lipoprotein complexes, are administered by injection,by a subcutaneously implantable pump or by a depot preparation, inamounts that achieve a circulating serum concentration equal to thatobtained through parenteral administration. The complexes could also beabsorbed in, for example, a stent or other device.

Administration can be achieved through a variety of different treatmentregimens. For example, several intravenous injections can beadministered periodically during a single day, with the cumulative totalvolume of the injections not reaching the daily toxic dose. The methodscomprise administering the lipoprotein complex at an interval of 6, 7,8, 9, 10, 11, or 12 days. In some embodiments, the lipoprotein complexis administered at an interval of a week.

The methods can further comprise administering the lipoprotein complex4, 5, 6, 7, 8, 9, 10, 11, or 12 times at any of the intervals describedabove. For example, in one embodiment, the lipoprotein complex isadministered six times, with an interval of 1 week between eachadministration. In some embodiments, administration could be done as aseries of injections and then stopped for 6 months to 1 year, and thenanother series started. Maintenance series of injections could then beadministered every year or every 3 to 5 years. The series of injectionscould be done over a day (perfusion to maintain a specified plasma levelof complexes), several days (e.g., four injections over a period ofeight days) or several weeks (e.g., four injections over a period offour weeks), and then restarted after six months to a year. For chronicconditions, administration could be carried out on an ongoing basis.Optionally, the methods can be preceded by an induction phase, when thelipoprotein complexes are administered more frequently.

In yet another alternative, an escalating dose can be administered,starting with about 1 to 5 doses at a dose between (50-200 mg) peradministration, then followed by repeated doses of between 200 mg and 1g per administration. Depending on the needs of the patient,administration can be by slow infusion with a duration of more than onehour, by rapid infusion of one hour or less, or by a single bolusinjection.

Toxicity and therapeutic efficacy of the various lipoprotein complexescan be determined using standard pharmaceutical procedures in cellculture or experimental animals for determining the LD50 (the doselethal to 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Lipoprotein complexes, such as negatively chargedlipoprotein complexes, that exhibit large therapeutic indices arepreferred. Non-limiting examples of parameters that can be followedinclude liver function transaminases (no more than 2× normal baselinelevels). This is an indication that too much cholesterol is brought tothe liver and cannot assimilate such an amount. The effect on red bloodcells could also be monitored, as mobilization of cholesterol from redblood cells causes them to become fragile, or affect their shape.

Patients can be treated from a few days to several weeks before amedical act (e.g., preventive treatment), or during or after a medicalact. Administration can be concomitant to or contemporaneous withanother invasive therapy, such as, angioplasty, carotid ablation,rotoblader or organ transplant (e.g., heart, kidney, liver, etc.).

In certain embodiments, negatively charged lipoprotein complexes areadministered to a patient whose cholesterol synthesis is controlled by astatin or a cholesterol synthesis inhibitor. In other embodiments,negatively charged lipoprotein complexes are administered to a patientundergoing treatment with a binding resin, e.g., a semi-synthetic resinsuch as cholestyramine, or with a fiber, e.g., plant fiber, to trap bilesalts and cholesterol, to increase bile acid excretion and lower bloodcholesterol concentrations.

6.8. Other Uses

The lipoprotein complexes, e.g., negatively charged lipoproteincomplexes, and compositions described herein can be used in assays invitro to measure serum HDL, e.g., for diagnostic purposes. BecauseApoA-I, ApoA-II and Apo peptides associate with the HDL component ofserum, negatively charged lipoprotein complexes can be used as “markers”for the HDL population, and the pre-beta1 and pre-beta2 HDL populations.Moreover, the negatively charged lipoprotein complexes can be used asmarkers for the subpopulation of HDL that are effective in RCT. To thisend, negatively charged lipoprotein complexes can be added to or mixedwith a patient serum sample; after an appropriate incubation time, theHDL component can be assayed by detecting the incorporated negativelycharged lipoprotein complexes. This can be accomplished using labelednegatively charged lipoprotein complexes (e.g., radiolabels, fluorescentlabels, enzyme labels, dyes, etc.), or by immunoassays using antibodies(or antibody fragments) specific for negatively charged lipoproteincomplexes.

Alternatively, labeled negatively charged lipoprotein complexes can beused in imaging procedures (e.g., CAT scans, MRI scans) to visualize thecirculatory system, or to monitor RCT, or to visualize accumulation ofHDL at fatty streaks, atherosclerotic lesions, and the like, where theHDL should be active in cholesterol efflux.

Examples and data associated with the preparation and characterizationof certain proApoA-I-lipid complexes are described in U.S. PatentPublication No. 2004/0067873, the disclosure of which is incorporatedherein by reference in its entirety.

Data obtained in an animal model system using certain proApoA-I-lipidcomplexes are described in U.S. Patent Publication No. 2004/0067873, thedisclosure of which is incorporated herein by reference in its entirety.

7. EXAMPLE 1 Development of an ApoA-I Expression System 7.1. Cloning andExpression of Human ApoA-I in Chinese Hamster Ovary (CHO) Cells 7.1.1.Preparation of the ApoA-I Expression Vector

The preproApoA-I gene sequence was obtained from NCBI (P02647) andflanking sequences were added for easy cloning and improved expression.The preproApoA-I encoding DNA with the flanking sequences wassynthesized and cloned into Bluescript KS+ vector. The 5′ flankingsequence contained an optimized Kozak translation sequence. ThepreproApoA-I insert was excised from the Bluescript KS+ vector with HindIII and Bgl II restriction enzymes. This fragment was gel-purified andligated into a retroviral expression vector bearing the followinggenetic elements: human cytomegalovirus promoter fused to a MoloneyMurine Sarcoma virus 5′ LTR, a MoMuLV/SV packaging region, theimmediate-early simian cytomegalovirus promoter, a multicloning site,the 3′ LTR from MoMuLV, and a bacterial origin of replication andbeta-lactamase gene. Clones of the resulting construct were sequencedthrough the gene and flanking regions. A final clone (clone #17) wasselected based on congruity to the predicted DNA sequence. Retrovectorwas then prepared for transduction using 293GP cells co-transfected withan expression plasmid for Vesicular Stomatitis Virus envelopeglycoprotein. Supernatant recovered from the co-transfected cells andconcentrated was used for the CHO-S transduction step described below inSection 7.1.2.

7.1.2. Production of a Mammalian Cell Line for Expression of ApoA-I

Chinese hamster ovary cells adapted for growth in serum-free medium(CHO-S) were subjected to three rounds of transduction (1×, 2×, and 3×)using the retroviral vector described above in Section 7.1.1. The pooledpopulation was expanded for cryopreservation after each transduction anda sample of cells was submitted for gene copy analysis. Gene copy indexis shown below in Table 2. The 3× transduced cell line was analyzed forproductivity in a 16-day fed batch test in duplication 125 mL flasks.The results are shown in Table 3.

TABLE 2 Gene Copy Index Results of ApoA-I Expression Cell Lines GeneCopy Transduction Round Cell Line Name After Transduction Index 1 *CHO-S-ApoA-I-R (1X) 2.53 1 * CHO-S-ApoA-I-R (1X) 2 ** CHO-S-ApoA-I-R(2X) 5.50 2 ** CHO-S-ApoA-I-R (2X) 3 * CHO-S-ApoA-I-R (3X) 7.23 * The 1Xcell lines were combined before proceeding to create the 2X cell lines** The 2X cell lines were combined before proceeding to create the 3Xcell lines

TABLE 3 Fed Batch Productivity Data for CHO-S-ApoA-I-R (3X) 3X PooledDay Population 2 4 6 8 10 12 VCD* 10.49 ** 66.33 69.10 74.73 51.74 (×105cells/mL) μg/mL (by ELISA) 89 459 1339 2755 2775 2658 *Viable celldensity **d4 cell counts not performed due to collection error

7.1.3. Stability of Cell Line Expressing ApoA-I

A non-GMP characterization study was conducted on CHO-S-ApoA-I-R (3×)clone, 17, the master cell bank producing ApoA-I, in order to assess itsstability in viability, growth rate, conservation of the gene inserts,and consistent product secretion during long-term culture.

The cell line was thawed from the master cell bank and cultured in 125mL shake flasks using PF CHO LS medium (HyClone, Logan Utah). The cellswere continuously cultured by serial passage from generation 0 togeneration 43. At generations 4, 8, 14, 19, 25, 31, 36 and 43 samples ofcells were frozen. At the end of the culturing, samples of cells fromall generations were thawed and used to conduct terminal culture runs tocompare ApoA-I protein production of the cell line at differentgenerations in the same experiment. The supernatant from each of thesamples were tested at day 12 using reverse phase HPLC analysis todetermine the level of ApoA-I production. ApoA-I concentration was foundto vary from 1259 mg/L to 1400 mg/L for the various samples (FIG. 2).

To compare the stability of gene inserts, samples of the CHO cells atgenerations 0, 4, 14, 25, 36 and 43 were used for DNA isolation. Thenumber of genetic inserts for each sample was determined using real-timePCR on genomic DNA. As shown in Table 3, the PCR-based indexes of copynumber were not significantly different based on overlapping standarddeviations between generation 0 and generation 43. The production ofApoA-I and the gene copy index values from the master cell bank cellline were found to be stable over the 43 generations tested.

TABLE 4 Stability of Gene Copy Index Generation Gene Copy Index StandardDeviation 0 7.87 0.25 4 7.80 0.27 14 7.80 0.27 25 7.97 0.25 36 7.90 0.1743 8.20 0.27

7.2. Cell Growth and Harvest of ApoA-I 7.2.1. Scale Up to Innoculum and200 L Bioreactor Production

A vial of CHO-S cells stably transfected with the human ApoA-I gene fromthe master cell bank were thawed in a 37° C. water bath and were addedto a single shake flask (250 mL) (Thermo Fisher Scientific) containing35 mL of HyQ PF-CHO LS cell culture medium. The initial cell count,determined using a hemacytometer, was 2.48×10⁵ cells/mL and 93.8%viability. The culture was then placed on an Orbit shaker (90 rpm)within an incubator maintained at 37° C. in a humidified, 5% CO₂environment. Subculture steps targeted inoculation densities ofapproximately 1.6×10⁵ cells/mL. The day 4 cell count and percentviability in the flask was 15.01×10⁵ cells/mL with 93.9% viability. The250 mL flask was subcultured to 1 L flask. The day 3 average viable cellcount from the 1 L flask was 13.56×10⁵ cells/mL with 95.3% viability.The 1 L flask was subcultured to a Wave Bioreactor System 20EH with a 10L disposable Wave Bag (GE Healthcare Bioscience Bioprocess Corp,Somerset, N.J.) at an initial culture volume and an initial target celldensity of 1000 mL and 1.75×10⁵ cells/mL, respectively. The WaveBioreactor operating settings were 37° C. incubation temperature, 15.0cpm rocker speed, 10.9° rocking angle, and 5.0% CO₂ concentration in theaeration gas with a gas flow rate of 0.25 L/minute. After 3 days ofculture, the viable cell density in the Wave Bag was 10.73×10⁵ cells/mL,and fresh HyQ PF-CHO LS was added bringing the culture volume to 5000mL. After an additional three days, the viable cell density was13.25×10⁵ cells/mL and the volume in the Wave Bag was transferred to the30 L bioreactor. The operating set-points in the 30 L bioreactor fortemperature, pH, dissolved oxygen, pressure, and agitation rate were 37°C., pH 7.0, 40% air saturation, 1 psig pressure, and 50 rpm agitationrate, respectively.

The viable cell density in the 30 L bioreactor was 10.80×10⁵ cells/mL onday three of culture, which was sufficient to inoculate the 200 Lbioreactor at an initial target density of 2.40×10⁵ cells/mL. The entirecontents of the 30 L were transferred and the post inoculum weight, celldensity and viability were respectively 134.5 Kg, 2.37×10⁵ cells/mL and90.9%. The operating set-points in the 200 L bioreactor for temperature,pH, dissolved oxygen, pressure, and agitation rate were 37° C., pH 7.0,40% air saturation, 1 psig pressure, and 35 rpm agitation rate,respectively. On day 3, the cell density was 15.71×10⁵ cells/mL, whichwas sufficient to add 60 L (v/v) of Complete Medium (AGT CD CHO 5×,Invitrogen) and 200 mM L-glutamine (final concentration 10 mM) solutionto the bioreactor. On days 8 and 12, the glucose level fell below 5 g/Lwhich triggered an addition of 3 g/L of glucose (20%) solution. Theviable cell density peaked on day 9 at 33.20×10⁵ cells/mL with aviability of 92.5%. The bioreactor was harvested on day 13 at a cellviability of 78.5%. The cell counts and viability of the culturethroughout the culture period are shown in FIG. 3A and the ApoA-Iconcentration in the culture medium throughout the culture period areshown in FIG. 3B.

7.2.2. Harvesting, Cell Separation and Storage of Cells

Media from the bioreactor was harvested by passing the bioreactorcontents through double Cuno (Rutherford, N.J., USA) 60MO2 Maximizerfilters followed by a Millipore 0.22 μm Opticap (Billercia, Mass., USA)into a 200 L bag. The clarified media was then stored at 2-8° C. untilthe dispensing operation. The clarified media was filtered through aMillipore 0.22 μm filter (Billerica, Mass., USA) and dispensed into 2 Lsterile PETG bottles (ThermoFisher, Marietta, Ohio, USA) and then frozenat −20° C. until released for shipment.

8. EXAMPLE 2 Development of an ApoA-I Purification System 8.1. Materialsand Methods

Expression, Primary Separation and Conditioning.

ApoA-I clarified cell growth medium obtained as described in Example 1(approximately 1.8 L) was thawed by storage at ambient temperature. Thethawed medium was conditioned for anion exchange chromatography byreducing the pH 5.3±0.2 with 1M HCl.

Purification of Apo A-I.

A Q-Sepharose FF (GE Healthcare) anion exchange column packed at a bedheight of 20 cm was equilibrated with TAMP A buffer (20 mM sodiumphosphate, pH 5.3). Equilibration was judged to be complete when the ofthe column effluent was approximately 5.5. The conditioned filtrate wasloaded onto the column at 25-35 g ApoA-I/L of anion exchange resin at aflow rate of 3.7 cm/min. ApoA-I was washed through the column in theTAMP A flow-through, which was collected.

The ApoA-I containing flow-through from the anion-exchange column was pHadjusted to 8.0±0.2 with 1M NaOH. The solution was then filtered througha 0.2 μm Planova 20N filter (Asahi Kasei Medical) at a flow rate ofabout 12.5 L/h/m² to remove viruses and viral particles.

A Source 30 reverse phase chromatograpy column with a bed height of 25cm was washed with TAMP D buffer (20 mM ammonium carbonate, pH 9.5)until equilibrated, when the column effluent reached pH 9.5. The ApoA-Icontaining filtrate from the virus filtration step was loaded onto thecolumn at a flow rate of 2.8 cm/min. ApoA-I in the sample was adsorbedto the matrix and eluted with a gradient of 35-50% acetonitrile in TAMPD buffer.

A reverse phase silica C18 column (300 Å 10 μm) packed at a bed heightof 25 cm was washed in TAMP E buffer (100 mM ammonium carbonate, pH 9.5)until equilibrated, when the column effluent reached pH 9.5. The C18column was then loaded at 4.7 g ApoA-I/L of matrix. The ApoA-I adsorbedto the column matrix was eluted in a 40-50% acetonitrile gradient inTAMP E buffer.

Acetonitrile was removed from the ApoA-I containing fractions elutedfrom the C18 column by pooling and concentrating the fractionsapproximately 2.5-fold and then diafiltering the concentration against15 volumes of TAMP C buffer (3 mM sodium phosphate, pH 8). The pH of thediafiltered ApoA-I solution was decreased to about 6.0 using dilutephosphoric acid and then passed through a Mustang Q anion exchangemembrane (Pall Life Sciences) to remove DNA and host cell proteins. TheMustang Q filtrate was diafiltered against 5 volumes of TAMP C buffer.

The diafiltered filtrate was then subjected to a final ultrafiltrationusing a polyethersulphone membrane (Filtron Omega series) with a 10,000dalton molecular weight cutoff so that the membrane retains the 28,000dalton ApoA-I. The protein solution contained The protein solutioncontained 7.8-17 g/L pure ApoA-I as determined by scanning an SDS-PAGEgel and measuring the ratio of the intensity of the purified ApoA-I bandarea and the total intensity of all bands.

8.2. Results

Characterization of ApoA-I.

The purity of the ApoA-I product was assayed by SDS-PAGE to be greaterthan 99% pure, with low levels of DNA and host cell proteins, and nodetectable amount of truncated ApoA-I. See FIG. 4.

9. EXAMPLE 3 Optimization of Lipoprotein Complex Components 9.1.Preparation of Apolipoprotein and Phospholipid Components

proApoA-I:

The protein proApoA-I was supplied by Unité de Biotechnologie, InstitutMeurice, Hte Ecole Lucia De Brouckère, 1 Avenue Emile Gryzon, B-1070Anderlecht, Belgium in lyophilized individual 100 mL flasks containingapproximately 90 mg of protein. The batch number was 20060202. Theprotein was kept at approximately 4° C. until use. Beforelyophilization, the content of proApoA-I was 3.225 mg/mL with an ureacontent about 0.011 mg/mL. A solution of proApoA-I was made bydissolving approximately 630 mg of proApoA-I in 25.6 mL of aceticacid/water 5%. The final concentration of the solution was 25 mg/mL.

ApoA-I:

ApoA-I was prepared as described in Example 1 above.

Sphingomyelin:

Sphingomyelin from egg (Coatsome® NM-10) was supplied by NOFCorporation, 1-56, Oohama-Cho, Amagasaki-Shi, 660-0095, Japan. The batchnumber was 0502ES1. Sphingomyelin was kept at approximately −20° C.until use. The purity of sphingomyelin was 99.1%. A solution ofsphingomyelin was made by dissolving 799.4 mg of purified sphingomyelinin 16 mL of acetic acid/water 5% to yield a final concentration of 50mg/mL.

Phosphatidylglycerol:

1,2-dipalmitoyl-SN-glycero-3-phosphatidyl glycerol as sodium salt(DPPG-Na, Coatsome® MG-6060 LS) was supplied by NOF Corporation, 1-56,Oohama-Cho, Amagasaki-Shi, 660-0095, Japan. The batch number was0309651L. DPPG-Na was kept at approximately −20° C. until use. Thepurity of DPPG-Na was 99.2%. A solution of DPPG-Na was made bydissolving 49.1 mg of DPPG-Na in 1 mL acetic acid/water 5% to yield afinal concentration of 50 mg/mL.

Phosphatidylcholine:

di-palmitoyl phosphatidylcholine (DPPC) was obtained from a commercialsource.

9.2. Preparation Of Lipoprotein Complexes

The following lipoprotein complexes were prepared:

(a) Neutral Lipoprotein Complexes:

-   -   a. Formula A: proApoA-I and SM in a protein:phospholipid weight        ratio of 1:2.5;    -   b. Formula B: proApoA-I and SM in a protein:phospholipid weight        ratio of 1:2.7;    -   c. Formula C: proApoA-I and SM in a protein:phospholipid weight        ratio of 1:3.1;    -   d. Formula D: proApoA-I, SM, and DPPC in a 1:2.7 lipoprotein wt:        total phospholipid wt ratio with a SM:DPPC wt ratio of 50:50;    -   e. Formula E: ApoA-I and SM in a protein:phospholipid weight        ratio of 1:2.7.

(b) Negatively Charged Lipoprotein Complexes:

-   -   a. Formula F: proApoA-I, SM, DPPC and DPPG in a 1:2.7        lipoprotein wt: total phospholipid wt ratio with a SM:DPPC:DPPG        wt:wt ratio of 48:48:4;    -   b. Formula G: proApoA-I, SM, DPPC and DPPG in a 1:2.7        lipoprotein wt: total phospholipid wt ratio with a SM:DPPC: DPPG        wt:wt ratio of 73:23:4;    -   c. Formula H: ApoAI, SM, and DPPG in a 1:2.7 lipoprotein wt:        total phospholipid wt ratio with a SM:DPPG wt:wt ratio of 97:3;    -   d. Formula I: ApoAI, SM, and DPPG in a 1:3.0 lipoprotein wt:        total phospholipid wt ratio with a SM:DPPG wt:wt ratio of 97:3;    -   e. Formula J: ApoAI, SM, and DPPG in a 1:3.3 lipoprotein wt:        total phospholipid wt ratio with a SM:DPPG wt:wt ratio of 97:3;

9.3. Rate of Formation and Homogeneity of Lipoprotein Complexes

Formation of lipoprotein complexes of Formulas A through J was tested byinjecting a sample into a HPLC system to check for the size anddistribution of lipoprotein complexes. Complexes were produced byco-homogenization and sampled at the indicated times.

FIG. 5 shows exemplary HPLC chromatograms for neutral lipoproteincomplexes according to Formulas A through C comprising differentlipoprotein:SM wt:wt ratios, and Formula D comprising lipoprotein andneutral phospholipids in a 1:2.7 ratio, where the neutral phospholipidis 50:50 SM:DPPC. The lipoprotein:SM wt:wt ratio of 1:2.7 was optimal.Formula D, which contained a mix of SM and DPPC, showed poor complexformation.

Addition of phosphatidyl choline as a second neutral phospholipidresulted in slow and incomplete complex formation. FIG. 6 shows HPLCchromatograms of lipoprotein complexes of Formula D at 10, 20, 30, and60 minutes. In contrast, as shown in FIG. 7, Formula B rapidly formedcomplexes.

Addition of a negatively charged phospholipid, DPPG, to SM and DPPC ledto even less complex formation, as shown in the FIG. 8 HPLCchromatograms of lipoprotein complexes of Formula F at 20, 40, 60, and120 minutes.

As shown in FIG. 9, lipoprotein complexes which contain only SM as aphospholipid, in a protein to lipid weight ratio of 1:2.7 form morepre-B HDL complexes and do so faster than lipoprotein complexes with thesame protein to lipid weight ratio but that contain DPPC and/or DPPG.Therefore, lipoprotein complexes comprising only SM as a neutral lipidform more homogeneous lipoprotein complexes at a faster rate thancomplexes comprising DPPC in addition to SM, with or without addition ofDPPG.

Finally, negatively charged lipoprotein complexes comprising anapolipoprotein: phospholipid weight ratio of between 1:2.7 and 1:3, inwhich the phospholipid fraction contains SM and DPPG in a 97 to 3 weightratio showed optimal homogeneity and no free lipid peak, as compared tocomplexes comprising an apolipoprotein: phospholipid weight ratio of1:3.3 and uncharged lipoprotein complexes. See FIG. 10, showing HPLCchromatograms for Formulae E, H, I, and J. Lipoprotein complexesaccording to Formulae B and H were chosen for further study in animals(Example 6) and, based on the results of the animal studies, lipoproteincomplexes of Formula H were chosen for clinical assessment in humanpatients (Examples 6 and 8).

10. EXAMPLE 4 Formation of Lipoprotein Complexes Using ThermalCycling-Based Methods 10.1. Overview of Procedure for MakingApoA-I/DPPG/Sphingomyelin Complexes

Frozen ApoA-I solution in phosphate buffer (pH 7-9) at a proteinconcentration of 1 to 30 mg/ml, typically 5 to 20 mg/ml, is prepared bythawing for approximately 24-96 hrs at 2-8° C. and weighed. Sodiummonobasic phosphate and sodium dibasic phosphate are added to the ApoA-Isolution to obtain a final peptide concentration of 10 mM in pH 7.4buffer.

DPPG solution is prepared by warming phosphate buffer (10 mM sodiumphosphate, pH 8.0) to a target of 50° C. DPPG powder (NOF Corporation)is thawed at ambient temperature for at least 1.5 hrs and then weighedand added to the buffer container. The DPPG is then dispersed attemperature of 50° C. using an ULTRA-TURRAX® high-performance disperser(IKA® Works, Inc.). After dispersion, the DPPG suspension and ApoA-Isolution are heated to 57° C. They are combined and heated at 57° C. for30 minutes under nitrogen. This pre-complex solution is cooled to roomtemperature.

Sphingomyelin (SM) powder (NOF Corporation) is thawed at ambienttemperature then weighed into a glass tank. The phosphate phosphatebuffer (10 mM sodium phosphate, pH 8.0) is heated to 50° C. combinedwith the SM powder for a SM concentration of 220 mg/ml. The SM powder isdispersed in suspension using an ULTRA-TURRAX® and the dispersion iscooled to 4° C. and then passed through the homogenizer. The SMparticles are monitored by dynamic light scattering (DLS) to 55 to 70 nmzeta (Z) average size (using an intensity measurement). This can beachieved, e.g., using a Nano DeBee homogenizer at 32000+/−3000 bars,with the temperature at the inlet at 10-18° C. and temperature at theoutlet preferably at 30-40° C. (and not exceeding 59° C.), resulting ina particle with Z-average size of 58 nm.

For complexation, the ApoA-I/DPPG mixture and the SM dispersion arewarmed separately to 57° C. The warmed SM dispersion is added to theApoA-I/DPPG mixture with an initial temperature set point of 57° C.After stirring to combine, the solution is cooled to 37° C. and thencarried through a series of thermal cycles (57° C. to 37° C.) in orderto form ApoA-I/DPPG/SM complexes. This heat-cool process is continuedwith a contact time between temperatures is from 5 minutes and 30minutes. The heat-cool cycles are repeated until the majority of theprotein component is incorporated into lipoprotein complexes. The sizeand distribution of complexes during thermocycling is monitored by gelpermeation chromatography (GPC).

ApoA-I/DPPG/SM complexes were made according to the procedure describedabove (and illustrated in FIG. 11). The ApoA-I protein had an amino acidsequence corresponding to positions 25 to 267 of the sequence depictedin FIG. 1. The complexes contain sphingomyelin (SM), and1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(Dipalmitoylphosphatidylglycerol or DPPG) in a 97:3 weight ratio. Theratio of ApoA-I protein-to-total lipids is 1:2.7 weight/weight (w/w)which is equivalent to a molar ratio of 1:108. The combined lipid andprotein components were cycled between 57° C. and 37° C. for 5 minutesat each temperature using a heat exchanger arranged as depicted in FIG.5, which includes on the left a Lauda Ecoline Star edition Type 26LEwater bath (in which the sample is thermally cycled) and on the right ashell & tube heat exchanger (model EF-050-HE), which has an 18-ml holdvolume, connected by a peristaltic pump.

180 mg of DPPG was added to 2.82 grams of 10 mM phosphate buffer pH 8.0.The suspension was dispersed with an ULTRA-TURRAX® disperser for 10minutes at 50° C. 22 grams of SM powder were combined with 78 grams of10 mM phosphate buffer pH 8.0. The suspension was dispersed with anULTRA-TURRAX® disperser for 20 to 40 minutes at 50° C. The SM washomogenized using a NanoBEE set to an average particle size of 60 nm.The DPPG, SM and protein were brought to 57° C. 18 mgs (0.3 ml) of DPPGwere added to 214 mg of ApoA-I protein in 10 mM phosphate buffer pH 7.4at 57° C. After 30 minutes at 57° C., 559 mg of SM particles (2.54 ml)were added. This solution was then subjected to thermal cyling to form acomplex.

Gel permeation chromatography showing ApoA-I/DPPG/SM complex formationafter thermocycling for 30 minutes, 60 minutes, 120 minutes, 180 minutesand 210 minutes is shown in FIGS. 13A-13E, respectively. A more compactcomplex is produced with increasing cycle time, as shown by theincreasing sharpness of the major GPC peak. The peak corresponding touncomplexed protein also disappears over time.

10.2. Formation of ApoA-I/Sphingomyelin Complexes

ApoA-I/SM complexes were made according to the procedure describedabove, but without pre-complexing ApoA-I with DPPG. The proteincomponent was 5 ml of ApoA-I at a concentration of 8.9 mg/ml and thelipid component was 0.5 ml egg sphingomyelin (220 mg/ml) which had beensuspended in 10 mM phosphate buffer pH 8.0 and homogenized to form lipidparticles of 60 nm. The protein and lipid components were mixed at 50°C. at a ratio of 1:2.5, wt:wt. The resulting suspension was subject tothermal cycling for 18 hours (108 cycles and cycling time of 10 minutes)using a thermal cycling apparatus as depicted in FIG. 5. Gel permeationchromatography shows that the protein/lipid complex formed isessentially homogeneous (see GPC chromatogram of FIG. 14).

10.3. Formation ofApoA-I/DPPG/N-palmitoyl-4-hydroxysphinganine-1-phosphocholine(phytosphingomyelin) Complexes

ApoA-I/DPPG/phytosphingomyelin complexes were made according to theprocedure described above. The phytosphingomyelin particles werehomogenized to a size of about 183 nm (measured by DLS) and added to the7.8 g/L protein:DPPG mixture to achieve a final protein to lipid (SM andDPPG) ratio of 1:2.7. The suspension containingN-palmitoyl-4-hydroxysphinganine-1-phosphocholine (phyto-sphingomyelin)and a protein:DPPG component was thermally cycled for six cycles of 10minutes at 37° C. and 10 minutes at 57° C., over a total of two hours.Gel permeation chromatography shows that the protein/lipid complexformed is essentially homogeneous (see GPC chromatogram of FIG. 15).

10.4. Formation of ApoA-I/DPPG/Synthetic Palmitoyl Sphingomyelin

ApoA-I/DPPG/synthetic palmitoyl sphingomyelin complexes were made asfollows. An 8.8 mL solution of synthetic palmitoyl sphingomyelin (220mg/ml) in 10 mM phosphate buffer pH 7.4 was mixed until a particle sizeof 3300 nm was reached. ApoA-I (945 mg at 14 mg/mi) was combined with0.03% DPPG (60 mg/ml) by weight and heated at 50° C. for 30 minutes. Thesynthetic palmitoyl sphingomyelin micelles were combined with theprotein/DPPG complex at an apolipoprotein:phospholipid weight ratio of1:2.7. The suspension of protein and lipid was thermally cycled withheat-cool cycles at 37° C. and 57° C. alternatively every ten minutesfor a total of 240 minutes or until the appropriate distribution of theparticle size is attained. The size and distribution of complexes duringthermocycling was monitored by GPC. After complexation the concentrationwas brought to 8.0 mg ApoA-I/ml, then sucrose (40 mg/ml) and mannitol(20 mg/ml) were added to the complex for isotonicity. The lipoproteincomplexes were assayed by GPC and found to be essentially homogenous(see GPC chromatogram of FIG. 16).

10.5. Formation of ApoA-I/DPPG/Phytosphingomyelin

ApoA-I/DPPG/phytosphingomyelin complexes were made as follows. A 2.0 mLsolution of phytosphingomyelin (220 mg/ml) in 10 mM phosphate buffer pH7.4 was dispersed in an ULTRA-TURRAX® for 40 minutes at 50° C. until aparticle size of 990 nm was reached. ApoA-I (15.6 mg at a concentrationof 7.8 mg/ml) was combined with 0.03% DPPG (60 mg/ml) by weight andheated at 57° C. for 30 minutes. The phytosphingomyelin solution wascombined with the protein/DPPG mixture at an apolipoprotein:phospholipidweight ratio of 1:2.7. The suspension of protein and lipid was thenthermally cycled with heat-cool cycles at 37° C. and 57° C.alternatively every ten minutes for a total of 240 minutes or until theappropriate distribution of the particle size was attained. Thepopulation of lipoprotein complexes was measured by GPC and found to beabout 92.6% homogeneous (see GPC chromatogram in FIG. 17).

10.6. Complex Formation with an ApoA-I Peptide

Complexes of ApoA-I peptide, DPPG and sphingomyelin were generated asdescribed above (see Section 10.1), using an ApoA-I peptide(H-Lys-Leu-Lys-Gln-Lys⁵-Leu-Ala-Glu-Leu-Leu¹⁰-Glu-Asn-Leu-Leu-Glu¹⁵-Arg-Phe-Leu-Asp-Leu²⁰-Val-Inp²²-OH;SEQ ID NO:4) solution. The phospholipid component consists of eggsphingomyelin (SM), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(Dipalmitoylphosphatidylcholine, DPPC) and1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](Dipalmitoylphosphatidyl-glycerol, DPPG) in a 48.5:48.5:3 weight ratio.The ratio of peptide to total phospholipids complex is 1:2.5 (w/w). Thedrug complex is a solution of the CER-522 complex in phosphate bufferedsaline (12 mM sodium phosphate, 130 mM sodium chloride, pH 8.2). Thecomplex was formed by subjecting the starting solution to thermalcycling between 50° C. and 37° C. for two hours, flow rate of 1 ml/min.

Gel permeation chromatography shows that the protein/lipid complexformed is essentially homogeneous, with a vast majority of the proteinhaving been incorporated into lipoprotein complexes (see GPCchromatogram of FIG. 18).

10.7. Effect of Lipid Particle Size and Number of Thermal Cycles onComplex Formation

The effect of lipid particle size and the number of thermal cycles onparticle size was studies. Preparations of four different starting lipidparticle sizes (with zeta averages of 85 nm, 77 nm, 66 nm, and 60 nm)(FIGS. 19A through 19D, respectively) were generated by passing SMsolutions through the NanoBEE in single passes. Each pass was analyzedfor its zeta average. The lipid particle size decreased with each passand the lipid particles were collected when the desired size wasachieved. The four different lipid particle sizes were tested in thecomplex formation method of Section 10.1, for five or seven cycles of 3minutes at 37° C. and 10 minutes at 57° C.

When the lipid component is mixed with the protein component, theresulting suspension is cloudy. The cloudiness is reduced, and thesolution becomes more transparent, as complexes are formed.

After five cycles, the suspension of complexes produced from 60 nm lipidparticles was the most transparent. GPC chromatograms (FIGS. 20A through20D) show complete or almost complete complexation in all samples, asevidenced by the homogeneity of major peak. When starting with 66 nmlipid particles, there was no uncomplexed protein detectable by GPC inthe resulting suspension of lipoprotein complexes, indicating that allthe protein was complexed with lipid after five cycles, and theresulting complexes were 98% pure. After seven cycles, all foursuspensions became more transparent, showing an even greater extent ofcomplexation. The suspension made using 60 nm lipid particles appearedthe clearest.

In a separate study, SM particles of 450 nm and 40 nm were complexedwith ApoA-I and DPPG using the method described in Section 10.1. Most,but not all, ApoA-I was incorporated into lipoprotein complexes using450 nm SM particles as the lipid component, as shown in the GPCchromatogram of FIG. 21A (the 9.8 minute peak). A much smaller fractionof ApoA-I was incorporated into lipoprotein complexes using 40 nm SMparticles as the lipid component, as shown in the GPC chromatogram ofFIG. 21B (the 9.551 minute peak).

10.8. Effect of Starting Temperature on Complex Formation

The effect of the initial thermal cycling temperature on complexformation was studied. ApoA-I/DPPG/SM complexes were generated describedin Section 10.1, except that the lipid component and the proteincomponent were warmed to, and combined at, 37° C. instead of 57° C. AGPC chromatogram of the resulting complex is shown in FIG. 22.Substantially less of the protein component was incorporated intolipoprotein complexes than when thermal cycling was initiated at 57° C.,as evidenced by the relatively large protein peak (eluting 9.455 minutesin FIG. 22).

10.9. Commercial Production of Lipoprotein Complexes Using ThermalCycling Methods

For large scale commercial manufacturing, the methods of the disclosurecan be scaled up and optionally combined with a formulation step. Acommercial embodiment is depicted in FIG. 23. In this embodiment,following the thermocycling steps, the lipoprotein complex is diluted,mixed with one or more isotonicity agents (e.g., sucrose and/ormannitol), filtered, and aliquoted into vials. The contents of the vialscan be freeze-dried to prolong shelf-life of the resulting formulation.

ApoA-I/DPPG/SM complexes described in Section 10.1 were produced on a20-liter scale using DaBEE2000. This complexes were diluted withphosphate buffer (pH 7-8) and mixed with sucrose and mannitol to a finalformulation containing phosphate buffer 10 mM pH 7.4, 8 mg/ml ApoA-I, 4%(w/w) sucrose and 2% (w/w) mannitol.

10.10. Comparison of Lipoprotein Complexes Made by Thermal Cycling Vs.Co-Homogenization

ApoA-I/DPPG/SM complexes made by the thermal cycling methods disclosedherein were compared to complexes made by co-homogenization of the lipidand protein components. The purity of the complexes made by thermalcycling was improved to 97% compared to the co-homogenization asmeasured by gel permeation chromatography. Using SDS-PAGE, the complexmade by thermal cycling has an increased main band purity of 98% withless truncated protein bands present as compared to co-homogenizedcomplexes.

Oxidation of the ApoA-I is also reduced by the thermal cycling processas compared to co-homogenization. RP-HPLC (C18) shows two oxidationpeaks at RT 0.93 and 0.99 in the co-homogenized complexes that are notpresent in the thermal cycling process. The peptide map also shows areduction in the oxidation of methionine of ApoA-I at Met 112 and Met148 in the complexes produced by thermal cycling as compared to thecomplexes produced by co-homogenization.

A summary of the data is presented in Table 5 below.

TABLE 5 Results of ApoA-I/DPPG/SM Complexes Manufactured usingCo-Homogenization and Thermal Cycling Batch Results Co-homogenizationCo-homogenization Thermal Cycling Thermal Cycling Test Batch A Batch BBatch A Batch B Purity of Complexes 86% 93% 96.9% 97.5% by GPC Purity ofApoA-I by Band # % Band # % Band # % Band # % SDS-PAGE 1 0.3 1 1.4 2 5.42 1.7 2 1.2 3 3.4 main 95.7 main 98.1 main 98.1 main 89.5 4 1.3 4 1.3Purity of ApoA-I by RRT % RRT % RRT % RRT % HP-SEC 0.97 4.9 0.97 1.50.97 1.2 0.96 0.7 1.00 98.2 1.00 84.9 1.00 98.2 1.00 98.7 1.10 12.8 1.1013.6 1.10 0.6 1.11 0.6 Heterogeneity RRT % RRT % RRT % RRT % ApoA-I byRP-HPLC 0.93 6.7 0.93 0.9 0.93 N/A 0.93 N/A (C18) 0.94 N/A 0.94 0.5 0.941.3 0.94 1.1 0.96 1.4 0.96 1.0 0.96 1.4 0.96 1.1 0.97 0.7 0.97 2.6 0.973.5 0.97 3.2 0.99 53.1 0.99 N/A 0.99 N/A 0.99 N/A 1.00 38.1 1.00 94.71.00 93.5 1.00 94.4 Heterogeneity of M₁₁₂ ox = 73.1%^(a) M₁₁₂ ox =5.5%^(a) M₁₁₂ ox = 4.9%^(a) M₁₁₂ ox = 3.6%^(a) ApoA-I by Peptide M₁₄₈ ox= 4.4% M₁₄₈ ox = 1.0% M₁₄₈ ox = 1.5% M₁₄₈ ox = 0.9% Map/Trypsin, RP-UPLC with UV Fingerprint Table 5 Abbreviations: GPC = Gel PermeationChromatography Rt = Retention time; NMT = Not More Than; SDS-PAGE =Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; HP-SEC = HighPerformance-Size Exclusion Chromatography; RP-HPLC = Reverse Phase-HighPerformance Liquid Chromatography; RRT = Relative Retention Time;RP-UPLC = Reverse Phase-Ultra Performance Liquid Chromatography; M₁₁₂ ox= ApoA-I Residue 112 Methionine Oxidation; M₁₄₈ ox = ApoA-I Residue 148Methionine Oxidation.

10.11. Use of Inert Gas in Manufacturing Methods

ApoA-I is a delicate protein that is susceptible to chemical instability(e.g., oxidation). To enhance the stability of ApoA-I inApoA-I/SM/DPPC-containing pharmaceutical compositions, thepharmaceutical compositions were prepared (including the thermalcycling, filling and finishing steps) under an atmosphere of nitrogen,an inert gas. Below are the results of studies comparing ApoA-I/SM/DPPCcomplexes made by co-homogenization and ApoA-I/SM/DPPC complexes made bythermal cycling under nitrogen.

TABLE 6 Comparative Analysis of Oxidation Levels of Met₁₁₂ and Met₁₄₈ inApoA-I/SM/DPPC Complexes Made by Two Different Methods Batch BatchManufacturing No. Size Method Atmosphere Oxidation Levels 1 10 LCo-homogenization Air M₁₁₂ ox = 14.8% M₁₄₈ ox = 0.8% 2 9.6 LCo-homogenization Air M₁₁₂ ox = 64.3% M₁₄₈ ox = 2.5% 3 9.0 LCo-homogenization Air M₁₁₂ ox = 71.3% M₁₄₈ ox = 2.5% 4 19.9 LCo-homogenization Air M₁₁₂ ox = 38.0% M₁₄₈ ox = 0.9% 5 18.0 LCo-homogenization Air M₁₁₂ ox = 5.9% M₁₄₈ ox = 0.8% 6 17.4 LCo-homogenization Air M₁₁₂ ox = 73.1% M₁₄₈ ox = 4.4% 7 12.0 L²Co-homogenization Air M₁₁₂ ox = 5.5% M₁₄₈ ox = 1.0% 8 21.6 L ThermalCycling Nitrogen M₁₁₂ ox = 4.9% M₁₄₈ ox = 1.5% 9 16.7 L Thermal CyclingNitrogen M₁₁₂ ox = 3.6% M₁₄₈ ox = 0.9%

11. EXAMPLE 5 In Vitro Cholesterol Efflux Studies

The biological activity of ApoA-I-lipid complexes was studied in Fu5AHrat hepatoma cells, measuring ABCA1-mediated cholesterol efflux.

The Fu5AH rat hepatoma cells have high expression of the scavengerreceptor class B type I (SRB1), which facilitate the bidirectional fluxof cholesterol between the cells and mature HDL. This model provides aspecific assay for IIDL-mediated cholesterol efflux activity. The assaywas performed using a method described by Mweva et al., 2006,“Comparison of different cellular models measuring in vitro the wholehuman serum cholesterol efflux capacity,” Eur. J. Clin. Invest. 36,552-559. Fu5AH cells were labeled with ³H-cholesterol for 24 hours.Acceptor media for efflux were prepared for each test sample(ApoA-I/DPPG/palmitoyl SM, ApoA-I/DPPG/egg SM, or ApoA-I/DPPG/phytoSM at30, 20 and 10 μg/ml, diluted with MEM buffered with 25 mM HEPES) and forthe control samples (ApoA-I purified from human plasma (20 μg/mL), HDL₃,2% human serum, medium alone). Acceptor medium containing test orcontrol samples was added to the cells for 4 hours and cholesterol wasmeasured in efflux media and cell monolayers to determine the percent ofcholesterol released from the Fu5AH cells. Biological activity of thetest samples was calculated and is expressed as percent of cholesterolefflux relative to a Reference Standard of Formula H described above atthe same concentration as the lipoprotein complex in the test sample,which served as a positive experimental control. Results are shown belowin Table 7, and demonstrate that the lipoprotein complexes tested havesignificant biological activity as measured in the cholesterol effluxassay.

TABLE 7 Biological Activity (by Induced Cholesterol Efflux) in the Fu5AHCell-Based Assay % of SR-BI Mediated Reference Complex % Efflux 20 μg/mlStandard ApoA-I/DPPG/Palmitoyl 3.70 ± 0.09 92% SphingomyelinApoA-I/DPPG/Egg Sphingomyelin 3.61 ± 0.05 89%ApoA-I/DPPG/Phytosphingomyelin 2.24 ± 0.04 56%

12. EXAMPLE 6 Pharmacodynamic Study of Single, Low Dose Administrationof Formula B and Formula H Complexes in Rabbits

Normal rabbits received a single injection of: (a) a 5 mg/kg dose of apreparation of Formula B (neutral lipoprotein complexes containingApoA-I and SM in a 1:2.7 apolipoprotein: phospholipid weight ratio); (b)a 5 mg/kg dose of a preparation of Formula H (negatively chargedlipoprotein complexes containing ApoA-I, SM, and DPPG in a 1:2.7apolipoprotein: phospholipid weight ratio and a 97:3 SM:DPPG weightratio); or (c) a control preparation, containing the diluent for thelipoprotein complex preparations. Four rabbits were tested with each ofFormula B, Formula H and control.

Plasma levels of VLDL total cholesterol and triglycerides over time areshown in FIGS. 24 and 25. Plasma VLDL total cholesterol levels increasedless and returned to baseline faster in the animals treated with FormulaH (ApoA-I/DPPG/SM complexes) (b), than the levels in animals treatedwith Formula B (lipoprotein complexes of ApoA-I and SM). A similareffect was seen for triglyceride levels. This study showed that thetransient elevation in these levels was of shorter duration withApoA-I/DPPG/SM complexes complexes than with neutral lipoproteincomplexes. This result is consistent with the results of a Phase I studyof the lipoprotein complexes of Formula H in human subjects (describedin Example 8 below).

13. EXAMPLE 7 Comparative Pharmacodynamic Studies of Egg SM andSynthetic Palmitoyl SM

The pharmacodynamic effect of Apolipoprotein A-I (ApoA-I)/egg SM andApoA-I/synthetic palmitoylSM lipoprotein complexes intravenouslyinjected into rabbits was studied. Following injection of thelipoprotein complexes, changes in plasma lipid and lipoprotein levelswere measured.

Lipoprotein complexes of either ApoA-I/egg SM or ApoA-I/synthetic SMwere administered to rabbits at doses of 5 mg/kg or 20 mg/kg byintravenous infusion into the ear vein, at a rate of 1 mL/min. 4 animalsper group were studied. Plasma samples were taken before dosing,immediately following the end of the infusion, and 30 min, 45 min, 1 h,2 h, 4 h, 6 h, 8 h, 10 h and 30 h after the initiation of the infusion.Plasma samples were then analyzed using commercial enzymatic kits fortotal cholesterol, unesterified cholesterol, phospholipids and totaltriglycerides. ApoA1 was assayed in plasma samples using commercialELISA kits. The plasma samples were analyzed by GPC to determine totaland unesterified cholesterol profiles. For the 5 mg/kg dose treatments,the results were quantified based on the percent of the total area underthe curve for the chromatogram trace.

FIG. 26A-26D show the plasma levels of cholesterol, triglycerides,phospholipids and apoA-I over time in rabbits infused with 5 mg/kg and20 mg/kg of either ApoA-I/egg SM or ApoA-I/synthetic SM. Rapid andsignificant cholesterol mobilization was observed within 30 minutesafter initiation of infusions at both doses administered: Cholesterolmobilization peaked at 30 minutes after administration for the 5 mg/kgdose, and a large increase in cholesterol mobilization was observed atthe 20 mg/kg dose. At each dose tested, both formulations had similarprofiles for plasma triglycerides, plasma phospholipids and plasmarecombinant human apoA-I.

FIG. 27A-27C show the plasma HDL-total cholesterol levels, plasmaLDL-total cholesterol levels, and plasma VLDL-total cholesterol levels.The increase of HDL-total cholesterol for ApoA-I/egg SM andApoA-I/synthetic SM was similar (FIG. 27A). There was little variationin plasma LDL-C and VLDL-C levels, and the levels were not substantiallyaltered by injection of lipoprotein complexes of either formulation(FIG. 27B-27C).

The results of this study show that ApoA-I/egg SM and ApoA-I/syntheticSM lipoprotein complexes elicit similar responses in vivo.

14. EXAMPLE 8 Phase I Study of ApoA-I/DPPG/SM Complexes in HealthyDyslidemic Subjects 14.1. Materials and Methods

A Phase I clinical trial was conducted with lipoprotein complexes ofFormula H as a randomized, double-blind, placebo controlled, cross-over,single rising-dose study in healthy volunteers with an LDL/HDL ratiogreater than 3.0. The objectives of this Phase I study were to assessthe safety, tolerability, pharmacokinetics and pharmacodynamics of anegatively charged lipoprotein complex when administered as a singledose. Escalating doses of 0.25, 0.75, 2.0, 5.0, 10.0, 15.0, 30.0 and45.0 mg/kg were studied. Subjects received by infusion a sterilesolution containing lipoprotein complexes of ApoA-I (prepared asdescribed in Example 1), SM and DPPG (in protein: lipid weight ratio of1:2.7 and lipid composition of 97% SM/3% DPPG (w/w)) that had been madeby co-homogenization of the protein and lipid components. The sterilesolution was a 10 mM phosphate buffered solution of pH 8.0 containingmannitol and sucrose (4% (w/w) sucrose, 2% (w/w) mannitol) in additionto the lipoprotein complexes.

14.2. Results

Below are summarized the clinical findings from this Phase I study.

Total Cholesterol:

Mean plasma total cholesterol concentrations at each time point arepresented in Table 8 below:

TABLE 8 Mean Plasma Total Cholesterol Concentrations by Time FollowingSingle IV Administration Hours Postdose [Plasma Concentration (mg/dL)]Dose 0 0.5 1 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   178.8 176.5171.3 175.0 180.0 181.8 182.8 172.8 176.8 181.0 168.0 186.3 189.3 0.75mg/kg   199.5 193.5 192.3 193.8 210.5 200.3 196.5 190.5 189.8 184.0189.8 205.0 196.3  2 mg/kg 208.0 201.8 209.8 207.8 211.3 217.3 216.0209.5 198.0 191.5 189.0 196.0 194.8  5 mg/kg 180.8 175.5 178.5 185.5191.5 192.5 181.5 187.0 190.8 191.8 181.3 189.8 194.3 10 mg/kg 180.3176.3 181.8 190.5 195.8 189.3 186.8 182.0 191.5 192.8 198.0 193.5 185.815 mg/kg 183.5 187.0 197.0 207.3 214.8 217.8 197.0 185.5 194.8 189.8200.5 188.8 186.0 30 mg/kg 185.0 190.3 205.8 227.5 234.0 224.3 201.5187.5 170.8 167.3 177.0 182.5 198.3 45 mg/kg 208.5 208.3 220.5 244.3269.0 278.5 262.3 273.8 233.3 231.8 228.0 213.3 223.8 Placebo 192.1184.5 182.6 188.0 192.7 196.0 189.8 192.1 187.2 186.3 189.9 192.6 195.3

VLDL, LDL and HDL in Total Cholesterol:

Mean values for VLDL, LDL and HDL in total cholesterol are summarized bytime point and dose in Tables 9-11 below:

TABLE 9 Mean VLDL in Total Cholesterol Following Single IVAdministration Hours Postdose [Plasma Concentration (mg/dL)] Dose 0 0.51 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   24.21 23.74 22.73 24.2226.62 31.00 32.67 29.12 32.48 35.61 23.50 34.35 32.56 0.75 mg/kg   31.0529.29 29.14 32.38 39.74 41.67 40.93 36.12 36.15 36.69 31.86 38.40 34.33 2 mg/kg 27.38 22.87 23.47 27.11 30.04 36.83 37.74 31.09 26.34 25.6824.58 28.41 21.22  5 mg/kg 23.04 19.65 21.02 25.01 31.10 34.34 29.8528.88 25.02 25.14 18.80 23.64 17.97 10 mg/kg 28.38 24.77 23.33 28.5938.11 43.53 39.07 37.98 33.70 33.08 28.83 27.37 24.17 15 mg/kg 21.7419.67 19.14 23.04 32.01 42.13 40.74 34.65 31.76 29.44 27.43 22.41 25.0230 mg/kg 21.28 16.58 13.28 18.70 28.95 44.06 48.54 55.64 37.56 28.4021.00 20.66 23.45 45 mg/kg 37.83 32.12 30.69 28.23 38.68 57.31 64.2681.80 69.79 60.82 47.92 37.99 42.00 Placebo 28.03 26.07 25.46 27.8629.45 35.52 33.99 31.77 31.33 36.51 26.91 29.42 28.29

TABLE 10 Mean LDL in Total Cholesterol Following Single IVAdministration Hours Postdose [Plasma Concentration (mg/dL)] Dose 0 0.51 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   112.65 111.83 109.38 111.44114.16 112.18 112.08 107.74 107.68 107.79 105.48 111.69 110.26 0.75mg/kg   129.52 126.39 125.37 124.40 131.92 121.88 120.06 118.66 117.25110.64 117.03 125.19 122.05  2 mg/kg 131.94 130.18 135.08 131.64 133.43133.42 131.74 132.63 128.20 122.92 119.23 121.89 124.59  5 mg/kg 110.67108.14 107.32 111.83 113.90 113.99 109.25 114.09 121.08 121.28 113.18115.33 123.03 10 mg/kg 106.56 102.15 102.83 105.92 106.73 102.29 104.74103.52 115.08 117.14 123.52 121.62 116.29 15 mg/kg 116.42 113.70 114.80118.08 120.51 119.67 108.28 106.08 117.27 116.84 122.43 120.18 114.27 30mg/kg 121.08 114.75 111.66 118.46 116.37 104.48 91.34 82.22 92.19 100.90112.18 115.92 126.04 45 mg/kg 127.94 124.70 126.10 132.06 136.32 133.79125.62 126.61 115.32 124.94 133.93 130.10 136.17 Placebo 119.77 115.96114.93 116.55 119.00 117.02 113.91 118.22 115.28 112.96 118.17 116.60119.54

TABLE 11 Mean HDL in Total Cholesterol Following Single IVAdministration Hours Postdose [Plasma Concentration (mg/dL)] Dose 0 0.51 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   41.89 40.93 39.14 39.3439.23 38.56 37.99 35.89 36.59 37.60 39.02 40.22 46.51 0.75 mg/kg   38.9337.81 37.74 36.98 38.83 36.70 35.51 35.71 36.36 36.67 40.87 41.41 39.86 2 mg/kg 48.68 48.70 51.20 49.00 47.78 47.00 46.52 45.78 43.46 42.9045.19 45.70 48.93  5 mg/kg 47.04 47.71 50.16 48.65 46.50 44.17 42.4144.03 44.65 45.33 49.27 50.78 53.25 10 mg/kg 45.31 49.32 55.59 55.9850.91 43.43 42.94 40.50 42.72 42.53 45.65 44.51 45.29 15 mg/kg 45.3453.63 63.05 66.13 62.23 55.95 47.97 44.77 45.72 43.47 50.64 46.15 46.7130 mg/kg 42.64 58.91 80.81 90.34 88.67 75.72 61.62 49.64 41.01 37.9543.82 45.93 48.76 45 mg/kg 42.73 51.42 63.70 83.96 94.00 87.40 72.3665.34 48.14 45.99 46.15 45.16 45.58 Placebo 44.26 42.47 42.16 43.6244.28 43.43 41.91 42.07 40.61 40.06 44.85 46.57 47.48

Unesterified (Free) Cholesterol:

Mean plasma free (unesterified) cholesterol concentrations at each timepoint are presented in Table 12 below:

TABLE 12 Mean Plasma Free Cholesterol Concentrations by Time FollowingSingle IV Administration Hours Postdose [Plasma Concentration (mg/dL)]Dose 0 0.5 1 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   48.8 46.0 44.846.0 46.5 49.0 51.0 46.0 48.0 50.8 44.0 49.0 49.0 0.75 mg/kg   54.0 52.851.8 54.0 58.3 56.5 55.8 52.8 51.0 50.5 51.0 56.5 52.0  2 mg/kg 51.851.5 54.0 54.5 56.5 60.0 59.8 55.0 52.0 50.8 50.8 50.8 49.8  5 mg/kg46.5 45.8 48.5 52.3 55.5 56.8 53.0 52.5 49.5 49.8 45.0 47.3 48.0 10mg/kg 47.0 47.3 52.3 58.3 62.0 60.0 59.3 54.8 52.8 50.8 51.5 48.0 46.815 mg/kg 46.5 50.5 57.8 67.3 74.0 75.0 69.0 58.8 55.8 51.8 51.5 48.049.0 30 mg/kg 48.0 55.5 69.0 87.0 95.3 94.5 86.0 74.0 54.0 47.8 47.047.0 51.5 45 mg/kg 54.3 57.5 66.5 87.3 110.0 122.0 119.5 124.0 92.3 79.561.5 57.0 59.8 Placebo 49.6 47.5 47.3 48.6 49.7 52.3 50.5 49.8 48.5 48.749.2 50.8 50.7

VLDL, LDL and HDL in Free Cholesterol:

Mean values for VLDL, LDL and HDL in free cholesterol are summarized bytime point and dose in Tables 13-15 below:

TABLE 13 Mean VLDL in Free Cholesterol Following Single IVAdministration Hours Postdose [Plasma Concentration (mg/dL)] Dose 0 0.51 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   10.78 10.19 9.88 10.4211.08 13.69 15.43 12.22 14.57 16.84 10.81 15.05 14.17 0.75 mg/kg   11.1110.58 10.43 12.02 14.63 16.06 15.81 13.74 14.36 14.93 12.20 14.33 12.92 2 mg/kg 9.15 8.24 8.59 10.08 11.47 14.56 14.48 11.13 10.04 10.04 9.7410.75 7.54  5 mg/kg 7.05 6.06 6.58 8.61 11.69 13.39 10.56 9.88 7.83 8.165.56 8.11 5.49 10 mg/kg 10.24 8.66 8.71 11.29 16.31 19.18 16.38 14.8812.50 12.02 9.94 9.13 8.46 15 mg/kg 8.37 7.39 7.45 10.18 15.78 22.3622.22 15.53 13.26 12.44 10.47 8.20 10.38 30 mg/kg 6.93 5.19 4.52 7.2313.88 23.42 28.04 29.43 15.23 10.82 7.82 7.19 8.67 45 mg/kg 12.93 10.7510.49 11.37 18.11 31.75 39.30 49.87 35.60 27.75 19.20 14.62 16.06Placebo 10.07 9.48 9.39 10.19 10.86 13.78 13.18 11.66 11.87 13.04 9.9911.53 10.99

TABLE 14 Mean LDL in Free Cholesterol Following Single IV AdministrationHours Postdose [Plasma Concentration (mg/dL)] Dose 0 0.5 1 2 4 8 12 2448 72 168 336 504 0.25 mg/kg   29.81 28.22 27.37 28.11 28.09 27.75 27.8726.92 26.45 26.77 25.97 26.62 26.47 0.75 mg/kg   35.40 34.45 33.57 34.5735.95 33.29 32.73 32.20 30.10 28.61 30.89 34.30 31.50  2 mg/kg 32.7932.52 33.46 33.44 35.04 35.50 35.10 34.72 33.34 32.24 31.33 30.74 32.22 5 mg/kg 29.12 27.62 27.55 30.07 32.23 32.72 32.09 32.71 32.24 31.9729.05 28.55 31.61 10 mg/kg 27.84 25.49 25.07 28.49 31.15 30.85 32.8031.35 31.95 30.77 32.55 30.64 30.12 15 mg/kg 28.85 26.58 26.15 30.3534.87 36.44 33.71 33.11 33.07 30.62 30.42 30.75 29.15 30 mg/kg 32.4227.36 26.02 30.10 33.66 34.18 32.23 31.26 29.90 29.20 31.55 32.11 34.5645 mg/kg 33.10 31.36 32.31 35.69 42.10 46.54 47.40 52.93 44.68 41.6733.72 34.22 35.45 Placebo 31.01 29.73 29.47 29.64 30.05 29.85 28.7630.34 29.22 28.30 30.73 30.28 30.73

TABLE 15 Mean HDL in Free Cholesterol Following Single IV AdministrationHours Postdose [Plasma Concentration (mg/dL)] Dose 0 0.5 1 2 4 8 12 2448 72 168 336 504 0.25 mg/kg   8.16 7.59 7.50 7.47 7.33 7.56 7.70 6.866.97 7.14 7.21 7.33 8.36 0.75 mg/kg   7.49 7.73 7.75 7.41 7.67 7.14 7.226.80 6.54 6.96 7.92 7.87 7.59  2 mg/kg 9.82 10.74 11.95 10.97 9.99 9.9310.17 9.16 8.62 8.47 9.68 9.26 9.99  5 mg/kg 10.32 12.07 14.36 13.5711.59 10.64 10.35 9.91 9.43 9.62 10.38 10.59 10.90 10 mg/kg 8.92 13.1018.47 18.47 14.54 9.96 10.07 8.52 8.30 7.96 9.01 8.23 8.17 15 mg/kg 9.2716.53 24.15 26.71 23.35 16.20 13.07 10.11 9.42 8.70 10.61 9.06 9.47 30mg/kg 8.65 22.95 38.46 49.67 47.70 36.89 25.73 13.31 8.87 7.73 7.64 7.708.27 45 mg/kg 8.21 15.39 23.70 40.19 49.79 43.71 32.80 21.20 11.97 10.098.58 8.16 8.24 Placebo 8.49 8.30 8.39 8.76 8.75 8.71 8.53 7.82 7.41 7.388.51 8.94 8.94

Triglycerides:

Mean plasma triglyceride concentrations at each time point are presentedin Table 16 below:

TABLE 16 Mean Plasma Triglyceride Concentrations by Time FollowingSingle IV Administration Hours Postdose [Plasma Concentration (mg/dL)]Dose 0 0.5 1 2 4 8 12 24 48 72 168 336 504 0.25 mg/kg   124.5 123.5123.5 120.8 123.8 224.8 273.0 144.5 185.8 203.3 136.00 214.3 168.0 0.75mg/kg   125.5 127.3 131.5 133.5 138.0 191.8 205.0 148.8 163.3 184.0177.5 180.8 180.8  2 mg/kg 103.3 103.8 114.0 122.0 130.8 204.0 216.0120.5 117.0 113.8 122.5 135.8 102.8  5 mg/kg 90.0 84.0 90.3 108.5 127.0194.0 132.0 105.5 89.0 93.0 88.5 122.8 70.0 10 mg/kg 130.8 122.8 129.5154.8 193.8 248.8 229.0 171.8 158.0 159.5 127.5 120.8 107.3 15 mg/kg109.3 113.5 129.0 168.3 236.8 382.3 364.5 180.5 170.8 157.3 124.8 88.8132.0 30 mg/kg 88.5 88.8 101.8 141.3 226.8 391.0 456.5 320.5 175.3 138.594.8 72.8 96.3 45 mg/kg 156.5 159.0 169.0 212.0 328.3 592.8 745.3 705.8491.5 408.5 236.0 181.3 216.5 Placebo 120.8 115.5 115.8 119.3 123.0215.9 199.0 130.7 138.3 160.3 125.8 156.2 139.2

ApoA-I:

Change of subjects' baseline ApoA-I in mg/dL over time is shown in Table17 below. The maximum changes in plasma ApoA-I are bolded for each dose.

TABLE 17 Mean Changes in Plasma ApoA-I by Time Following Single IVAdministration Hours Post Start of Infusion Dose (mg/kg) 0.5 1 2 4 8 1224 0.25 −1.8 −3.9 −1.5 −1.1 −1.8 −1.5 −5.4 0.75 −0.9 −3.8 −2.5 −1.9 −0.7−5.2 −3.1 2 −2.3 −3.8 −2.1 −0.8 0.1 −3.7 −2.4 5 0.5 6.8 8.1 5.3 2.6 −2−0.6 10 8.6 17.8 17.8 13.9 7 5 0.3 15 16.3 32.5 30.9 27.4 21.3 10.3 8.530 36.1 71.8 68.3 61.8 45.8 24 15.4 45 23.3 48.6 93.8 93 71.3 39.2 14.9

Transaminase:

Mean values for liver alanine aminotransferase, or transaminase, levels,which are correlated with toxicity, are presented in Table 18 below. Thenormal range of alanine aminotransferase is 9 to 60 IU/L.

TABLE 18 Summary of Mean Values for Selected Alanine Aminotransferase byTreatment and Time Point Time Dose (mg/kg) Point Placebo 0.25 0.75 2.05.0 10.0 15.0 30.0 45.0 Base- 26.97 40.0 24.8 27.0 34.3 25.8 25.3 29.7524.50 line 12 h 25.84 40.8 21.0 25.0 33.5 26.5 25.3 35.25 33.25 24 h26.47 39.3 19.8 23.8 34.5 25.3 26.5 37.75 32.00 48 h 26.16 39.5 21.822.3 35.8 23.5 29.0 36.25 28.50 72 h 27.22 39.5 23.8 22.5 35.5 22.8 31.536.50 29.00  7 d 27.97 40.3 25.3 24.8 33.5 20.0 43.3 35.75 27.00 14 d30.72 41.8 26.3 25.5 37.5 21.5 25.8 32.00 24.00 21 d 29.13 49.7 25.027.0 32.3 22.3 34.3 28.50 23.25 Placebo 0.25 0.75 2.0 5.0 10.0 15.0 30.045.0

Adverse Events:

A total of 7 (22%) subjects had adverse events. No subjects had adverseevents considered by the investigator to be at least possibly related tostudy drug. There do not appear to be any dose-related trends in theoccurrence of adverse events. No subjects had a serious adverse event,and no subject withdrew from the study due to an adverse events. Table19 below provides a summary of adverse events by body system:

TABLE 19 Summary of Adverse Events by Body System [Number (%) ofSubjects] Dose of Complex Body System Placebo 0.25 mg/kg 0.75 mg/kg 2.0mg/kg 5.0 mg/kg 10 mg/kg 15 mg/kg 30 mg/kg 45 mg/kg Adverse event (N =32) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4) (N = 4)Gastrointestinal System Diarrhoea 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0(0%)  1 (25%) 0 (0%) 0 (0%) Abdominal 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)0 (0%) 0 (0%)  1 (25%) 0 (0%) Pain/Cramping Musculoskeletal System ToeInjury 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)  1 (25%)Infections and Infestations Stye 0 (0%) 0 (0%)  1 (25%) 0 (0%) 0 (0%) 0(0%) 0 (0%) 0 (0%) 0 (0%) Cold Symptoms 1 (3%) 0 (0%) 0 (0%) 0 (0%) 0(0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Nervous System Vasovagal Episode 1 (3%)0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) RespiratoryCough 1 (3%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)Sore Throat 1 (3%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0(0%)

14.3. Conclusions

This Phase I study, which administered a preparation of a sterilesolution comprising a negatively charged lipoprotein complex of FormulaH in single IV doses of 0.25, 0.75 2.0, 5.0, 10.0, 15.0, 30.0 and 45.0mg/kg, resulted in the following conclusions.

The preparation was well-tolerated at all doses in all subjects with anadverse event profile similar to that observed with placebo. The complexdoes not appear to affect clinical chemistry, hematology or coagulationparameters differently from placebo. No adverse effects on ECGs wereobserved. No antibodies to ApoA-I were detected following single doseadministration.

Plasma concentrations of ApoA-I and sphingomyelin increased with dose:ApoA-I levels returned to baseline by 24 hours post-dose for doses up to10 mg/kg and by 72 hours post-dose for doses above 10 mg/kg.Sphingomyelin levels returned to baseline by 24 hours post-dose fordoses up to 5 mg/kg, by 72 hours post-dose for doses from 10 to 30mg/kg, and by 7 days post-dose for subjects dosed with 45 mg/kg.

Cholesterol mobilization increased with increasing doses: Mobilizationin the HDL fraction of free cholesterol was seen with doses as low as2.0 mg/kg (mean 23% increase from baseline) and increased with dose.Triglyceride levels were transiently increased above levels seen withplacebo at doses of 15 mg/kg and above.

In addition, administration of the complex did not significantly raiseliver transaminase levels, and in all cases, the levels remained wellwithin the normal range. This is in contrast to CSL-111, a reconstitutedpurified human ApoA-I from plasma complexed with soybeanphosphtidylcholine, which has been seen to raise alanineamino-transferase levels up to more than 100-fold the upper normal limitin some patients, when administered at a dose of 80 mg/kg of patientweight. Tardif et al., 2007, JAMA 297:1675-1682.

Thus, the complexes of the disclosure can be administered at doses lowerthan those reported for other preparations that mimic HDL and stillachieve clinically meaningful improvements in lipid parameters withoutdetrimental side effects.

15. EXAMPLE 9 Phase II Clinical Study of Apoa-I/SM/DPPG Complexes in theTreatment of Subjects with Acute Coronary Syndrome

A clinical trial is conducted to further confirm the therapeutic benefitof low doses of negatively charged lipoprotein complexes of Formula H(ApoA-1, egg-sphingomyelin (egg-SM), and DPPG, in an apolipoprotein:phospholipid weight ratio of 1:2.7, with an egg-SM to DPPG weight ratioof 97:3) in the treatment of cardiovascular diseases. The ApoA-I isprepared by expression in CHO cells as described above in Examples 1,and the complexes are generated by the thermocycling methods of Example4.

Subjects presenting with symptoms of ACS are eligible to be screened forthis study. At the time of baseline catheterization, subjects need tohave an adequate intravascular ultrasound (IVUS) evaluation of one“target” artery for IVUS which is not influenced by prior or presentPCI, and the proximal 4 cm of the target artery should have a diameterstenosis between 0 and 50% by visual angiographic assessment, areference diameter ≧2.5 mm and be free of filling defects suggestive ofthrombus. Once the baseline IVUS has been evaluated by the IVUS CoreLaboratory for overall quality, the presence of a suitable target vesseland the absence of technical factors which can preclude accurate readingof the IVUS images, the subject is randomized to receive an intravenousinfusion, given over one hour, of placebo or one of three doses of thecomplexes (3, 6, or 12 mg/kg). Randomized subjects return at weeklyintervals (i.e., every 7 to 11 days) for five additional infusions.End-of-treatment labs are drawn one week (5 to 9 days) after the lastinfusion. A follow-up IVUS is conducted approximately 3 weeks (14 to 35days) after the last infusion. A follow-up visit occurs approximately 6months (+/−2 weeks) after the last infusion to collect samples forAnti-ApoA1 antibody testing and to monitor for major adverse cardiacevent (MACE) endpoints.

The primary endpoint is the nominal change in total plaque volume in a30 mm segment of the target coronary artery assessed bythree-dimensional IVUS. Other efficacy measurements include the percentchange in plaque volume and the change in percent atheroma volume in thetarget 30 mm segment, the change in total vessel volume in the target 30mm segment, as well as changes in plaque, lumen and total vessel volumesfrom baseline to follow-up in anatomically comparable 5 mm segmentscentered on the site with the smallest plaque burden at baseline, andthe largest plaque burden at baseline on three-dimensional IVUS. Thepercent change in plaque volume is calculated as the nominal changedivided by the baseline value, multiplied by 100. Percent atheroma(obstructive) volume is computed by dividing plaque volume by elasticexternal membrane (EEM) volume and then multiplying by 100.

16. SPECIFIC EMBODIMENTS Citation of References

Various aspects of the present disclosure are described in theembodiments set forth in the following numbered paragraphs.

1. A lipoprotein complex comprising an apolipoprotein fraction and alipid fraction, wherein said lipid fraction consists essentially of 95to 99 weight % neutral phospholipid and 1 to 5 weight % negativelycharged phospholipid, wherein the apolipoproteinfraction-to-phospholipid fraction ratio is in the range of about 1:2.7to about 1:3 by weight.2. The lipoprotein complex of embodiment 1 in which the apolipoproteinis selected from preproapoliprotein, preproApoA-I, proApoA I, ApoA-I,preproApoA-II, proApoA II, ApoA II, preproApoA-IV, proApoA-IV, ApoA-IV,ApoA-V, preproApoE, proApoE, ApoE, preproApoA I_(Milano),proApoA-I_(Milano), ApoA-I_(Milano), preproApoA-I_(Paris),proApoA-I_(Paris), and ApoA-I_(Paris) and mixtures thereof.3. The lipoprotein complex of embodiment 2 in which the apolipoproteinconsists essentially of ApoA I having at least 90% or at least 95%sequence identity to a protein corresponding to amino acids 25 to 267 ofSEQ ID NO:1.4. The lipoprotein complex of embodiment 3 in which the apolipoproteincomprises a monomer, dimer and/or tetramer.5. The lipoprotein complex of any one of embodiments 1 to 4 in which theapolipoprotein comprises an ApoA-I peptide mimetic.6. The lipoprotein complex of any one of embodiments 1 to 5, wherein theapolipoprotein fraction-to-phospholipid fraction ratio is 1:2.7 byweight.7. The lipoprotein complex of any one of embodiments 1 to 6, in whichthe lipid:apolipoprotein molar ratio ranges from about 1:105 to about110, where the apolipoprotein value is expressed in ApoA-I equivalents.8. The lipoprotein complex of embodiment 7, in which thelipid:apolipoprotein molar ratio is 1:108.9. The lipoprotein complex any one of embodiments 1 to 8, wherein theneutral lipid is natural sphingomyelin or synthetic sphingomyelin.10. The lipoprotein complex of embodiment 9 in which the sphingomyelinis egg-sphingomyelin.11. The lipoprotein complex of embodiment 9 which is made from asphingomyelin that is at least 95% pure.12. The lipoprotein complex of any one of the embodiments 1 to 11,wherein said lipid fraction consists essentially of 96 to 98 weight %neutral phospholipid and 2 to 4 weight % negatively chargedphospholipid.13. The lipoprotein complex of embodiment 12, wherein said lipidfraction consists essentially of 97 weight % neutral phospholipid and 3weight % negatively charged phospholipid.14. The lipoprotein complex of embodiment 13, in which the negativelycharged phospholipid comprises phosphatidylglycerol.15. The lipoprotein complex of embodiment 14 in which the negativelycharged phospholipid comprises or consists of a salt of1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG).16. The lipoprotein complex of embodiment 15 in which the salt is asodium, or potassium salt.17. The lipoprotein complex of any one of the embodiments 1 to 16 inwhich the acyl chains of the neutral and/or negatively chargedphospholipids are each, independently of one another, selected from asaturated or a mono-unsaturated hydrocarbon containing 12 to 26, 14 to26, or 16 to 26 carbon atoms.18. The lipoprotein complex of embodiment 17 in which each acyl chain ofthe neutral and/or negatively charged phospholipid are the same.19. The lipoprotein complex of embodiment 17 in which each acyl chain ofthe neutral and/or negatively charged phospholipid is different.20. The lipoprotein complex of embodiment 17 in which the acyl chains ofthe neutral and negatively charged phospholipid contain the same numberof carbon atoms.21. The lipoprotein complex of embodiment 17 in which the acyl chains ofthe neutral and negatively charged phospholipid have different degreesof saturation.22. The lipoprotein complex of embodiment 17 in which the acyl chains ofthe neutral and negatively charged phospholipid contain 16 carbon atoms.23. The population of lipoprotein complexes according to any one ofembodiments 1 to 22.24. A population of lipoprotein complexes, each comprising a lipidfraction and an apolipoprotein fraction consisting essentially of anapolipoprotein A-I (“ApoA-I”), wherein the population is characterizedby one, two, three, four, five, six, seven, eight, nine or all ten ofthe following characteristics:

-   -   (a) at least 80%, at least 85%, at least 90%, or at least 95% by        weight over ApoA-I in said population is in mature form;    -   (b) no more than 20%, no more than 15%, no more than 10% or no        more than 5% by weight of ApoA-I in said population is in        immature form;    -   (c) the population contains no more than 100 picograms, no more        than 50 picograms, no more than 25 picograms, no more than 10        picograms or no more than 5 picograms host cell DNA per        milligram of ApoA-I;    -   (d) the population contains no more than 500 nanograms, no more        than 200 nanograms, no more than 100 nanograms, no more than 50        nanograms, or no more than 20 nanograms host cell protein per        milligram of ApoA-I;    -   (e) no more than 20%, no more than 15%, no more than 10% or no        more than 5% by weight of ApoA-I in the population is in        truncated form;    -   (f) no more than 20%, no more than 15%, no more than 10%, no        more than 5%, no more than 3%, no more than 2% or no more than        1% of each of methionine 112 and methionine 148 in said ApoA-I        in said population is oxidized;    -   (g) at least 80%, at least 85%, at least 90% or at least 90% of        the lipoprotein complexes are in the form of particles of 4 nm        to 15 nm or 6 nm to 15 nm in size as measured by gel permeation        chromatography (“GPC”) or dynamic light scattering (“DLS”);    -   (h) the population contains no more than 1 EU, no more than 0.5        EU, no more than 0.3 EU or no more than 0.1 EU of endotoxin per        milligram of ApoA-I; and    -   (i) no more than 10%, no more than 5%, no more than 4%, no more        than 3%, no more than 2% or no more than 1% of the amino acids        in the ApoA-I in said population is deamidated.        25. The population of embodiment 24 in which no more than 15%,        or no more than 10%, no more than 5% or no more than 2% by        weight of the lipid in the lipid fraction in said complexes is        cholesterol.        26. The population of embodiment 25 which does not contain        cholesterol.        27. The population of any one of embodiments 24 to 26 in which        at least 85%, at least 90%, or at least 95% of the protein is        mature ApoA-I protein.        28. The population of embodiment 27 in which less than 15%, less        than 10%, or less than 10% of the protein is oxidized,        deamidated, and/or truncated species.        29. The population of any one of embodiments 24 to 28 in which        the lipoprotein complexes are at least 90%, at least 92.5%, at        least 95%, at least 96%, at least 97% or at least 98% pure.        30. The population of any one of embodiments 24 to 29 in which        the lipoprotein complexes are at least 80%, at least 85%, at        least 90% or at least 95% homogeneous, as reflected by a single        peak in gel permeation chromatography.        31. The population of embodiment 30 in which at least 80%, at        least 85%, at least 90% or at least 95% of the lipoprotein        complexes range 4 nm to 12 nm in size, 6 nm to 12 nm in size, or        8 nm to 12 nm in size, as measured by GPC or DLS.        32. The population of any one of embodiments 24 to 31 in which        at least 95%, at least 96%, at least 97%, at least 98% or at        least 99% of the protein is in complexes.        33. The population of any one of embodiments 24 to 32 which does        not contain cholate.        34. The population of any one of embodiments 24 to 33 which does        not contain any detergent.        35. The population of any one of embodiments 24 to 34 which        contains less than 200 ppm, less 100 ppm or less than 50 ppm of        non-aqueous solvent.        36. The population of any one of embodiments 24 to 35 wherein        said ApoA-I is a human ApoA-I protein.        37. The population of any one of embodiments 24 to 36 wherein        said ApoA-I is a recombinant ApoA-I.        38. The population of any one of embodiments 24 to 37 wherein        the ApoA-I has an amino acid sequence with at least 90% or at        least 95% sequence identity to a protein corresponding to amino        acids 25 to 267 of SEQ ID NO:1.        39. The population of any one of embodiments 24 to 38 wherein        said lipid fraction consists essentially of 95 to 99 weight %        neutral phospholipid and 1 to 5 weight % negatively charged        phospholipid.        40. The population embodiment 39 wherein said lipid fraction        consists essentially of 96 to 98 weight % neutral phospholipid        and 2 to 4 weight % negatively charged phospholipid.        41. The population of any one of embodiments 24 to 40, wherein        said lipid fraction consists essentially of 97 weight % neutral        phospholipid and 3 weight % negatively charged phospholipid        42. The population of embodiment 41, wherein the neutral lipid        is natural sphingomyelin or synthetic sphingomyelin, optionally        wherein the lipid has a peroxide value of less than 5 meq O/kg,        less than 4 meq O/kg, less than 3 meq O/kg, or less than 2 meq        O/kg.        43. The population of embodiment 42 in which the sphingomyelin        is egg-sphingomyelin.        44. The population of embodiment 42 which is made from a        sphingomyelin that is at least 95% pure.        45. The population of of any one of embodiments 41 to 44,        wherein the negatively charged phospholipid comprises        phosphatidylglycerol.        46. The population of embodiment 45 in which the negatively        charged phospholipid comprises or consists of a salt of        1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG).        47. The population of embodiment 46 in which the salt is a        sodium, or potassium salt.        48. The population of any one of embodiments 24 to 47 in which        the acyl chains of the neutral and/or negatively charged        phospholipids are each, independently of one another, selected        from a saturated or a mono-unsaturated hydrocarbon containing 12        to 26, 14 to 26, or 16 to 26 carbon atoms.        49. The population of embodiment 48 in which each acyl chain of        the neutral and/or negatively charged phospholipid are the same.        50. The population of embodiment 48 in which each acyl chain of        the neutral and/or negatively charged phospholipid is different.        51. The population of embodiment 48 in which the acyl chains of        the neutral and negatively charged phospholipid contain the same        number of carbon atoms.        52. The population of embodiment 48 in which the acyl chains of        the neutral and negatively charged phospholipid have different        degrees of saturation.        53. The population of embodiment 48 in which the acyl chains of        the neutral and negatively charged phospholipid contain 16        carbon atoms.        54. The population of any one of embodiments 24 to 41 which has        an apolipoprotein fraction:lipid fraction molar ratio ranging        from 1:80 to 120, from 1:85 to 1:110, or from 1:100 to 1:115,        where the apolipoprotein value is expressed in ApoA-I        equivalents.        55. The population of embodiment 54 which has an apolipoprotein        fraction:lipid fraction molar ratio ranging from 1:80 to 1:90,        from 1:90 to 1:100, from 1:100 to 1:110 or from 1:105 to 1:110,        where the apolipoprotein value is expressed in ApoA-I        equivalents.        56. The population of any one of embodiments 24 to 55 which has        an apolipoprotein fraction-to-phospholipid fraction ratio        ranging from 1:2 to about 1:3 by weight.        57. The population of any one of embodiments 24 to 40, wherein        the apolipoprotein fraction-to-phospholipid fraction ratio        ranges from 1:2.1 to 1:2.7 by weight.        58. The population of embodiment 57 wherein the apolipoprotein        fraction-to-phospholipid fraction ratio is 1:2.7 by weight.        59. A pharmaceutical composition comprising or consisting        essentially of a lipoprotein complex according to any one of        embodiments 1 to 22 or a population of lipoprotein complexes        according to any one of embodiments 23 to 58, and one or more        pharmaceutically acceptable carriers, diluents and/or        excipients.        60. A mammalian host cell engineered to express an ApoA-I        protein, said ApoA-I protein comprising an amino acid sequence        having at least 95% identity to positions 25 to 267 of SEQ ID        NO:1.        61. The mammalian host cell of embodiment 60, wherein the        protein is secreted into the medium when the host cell is        cultured.        62. The mammalian host cell of embodiment 60 or embodiment 61,        wherein the protein further comprises the signal sequence        MKAAVLTLAVLFLTGSQA.        63. The mammalian host cell of any one of embodiments 60 to 62,        wherein the protein further comprises the propeptide sequence        RHFWQQ.        64. The mammalian host cell according to any one of embodiments        60 to 63, which is Chinese hamster ovary (CHO), CHO-S, CHO-K1,        VERO, BHK, BHK 570, HeLa, COS-1, COS-7, MDCK cells, 293, 3T3,        myeloma, PC12 and W138.        65. The mammalian host cell according to embodiment 64, which is        a CHO cell. 66. The mammalian host cell according to embodiment        65, which is a CHO-S cell or a CHO-K1 cell.        67. The mammalian host cell according to any one of embodiments        60 to 66 which is capable of producing at least 0.5, 1, 2, 3, or        4 g/L of said ApoA-1 protein in culture.        68. The mammalian host cell according to embodiment 67, which is        capable of producing up to 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20        g/L of said ApoA-I protein in culture.        69. The mammalian host cell of embodiment 67 or embodiment 68,        wherein the culture is a large scale culture.        70. The mammalian host cell of embodiment 69 wherein said large        scale culture is at least 15 liters, at least 20 liters, at        least 25 liters, or at least 30 liters.        71. The mammalian host cell of embodiment 70, wherein said large        scale culture is about 50 liters, about 100 liters, about 200        liters or about 300 liters.        72. The mammalian host cell according to any one of embodiments        60 to 71, which comprises at least about 5 copies of a        nucleotide sequence encoding said ApoA-I protein.        73. The mammalian host cell according to embodiment 72, wherein        each nucleotide sequence is operably linked to a promoter.        74. The mammalian host cell according to embodiment 73, wherein        the promoter is a cytomegalovirus promoter.        75. The mammalian host cell according to embodiment 74, wherein        the promoter is an immediate early simian cytomegalovirus        promoter.        76. The mammalian host cell according to any one of embodiments        60 to 75 which secretes a mature ApoA-I protein comprising or        consisting of an amino amino sequence corresponding to amino        acids 25 to 267 of SEQ ID NO:1.        77. A mammalian cell culture comprising a plurality of the        mammalian host cell according to any one of embodiments 60 to        76.        78. The mammalian cell culture according to embodiment 77, which        comprises at least about 0.5 g/L of mature ApoA-I protein        comprising or consisting of an amino amino sequence        corresponding to amino acids 25 to 267 of SEQ ID NO:1.        79. The mammalian cell culture according to embodiment 78, in        which at least 80%, at least 85%, or at least 90% of said mature        ApoA-I protein lacks a signal sequence.        80. The mammalian cell culture according to embodiment 78, in        which at least 80%, at least 85%, or at least 90% of said mature        ApoA-I protein lacks a signal sequence and a propeptide        sequence.        81. The mammalian cell culture according to any one of        embodiments 78 to 80, in which at least 80%, at least 85% or at        least 90% of said mature ApoA-I protein is not truncated,        oxidized or deamidated.        82. A method of producing mature, biologically active ApoA-1        protein, comprising culturing the mammalian host cell according        any one of embodiments 60 to 76 under conditions in which the        ApoA-I protein is expressed and secreted.        83. The method of embodiment 82, further comprising recovering        from the supernatant of said cultured mammalian host cell said        mature, biologically active ApoA-1 protein.        84. The method according to embodiment 82 or embodiment 83,        further comprising purifying ApoA-I protein.        85. A pharmaceutical composition comprising a therapeutically        effective amount of an ApoA-I protein obtained or obtainable by        the method of embodiment 84.        86. The pharmaceutical composition of embodiment 85 in which the        ApoA-I protein is complexed with lipid.        87. A method for minimizing oxidation products in a        pharmaceutical composition comprising ApoA-I, comprising        manufacturing said pharmaceutical composition under an inert        gas.        88. The method of embodiment 87, wherein the inert gas is        nitrogen, helium or argon.        89. The method of embodiment 87 or embodiment 88, wherein the        pharmaceutical composition is a pharmaceutical composition of a        lipoprotein complex comprising ApoA-I.        90. A method for preparing lipoprotein complexes, comprising:    -   (a) cooling a starting suspension comprising a lipid component        and a protein component from a temperature in a first        temperature range to a temperature in a second temperature        range,    -   wherein said lipid component consists essentially of particles        of lipids and wherein said protein component consists        essentially of lipid-binding peptides and/or lipid-binding        proteins;    -   (b) heating the cooled suspension of (a) from a temperature in        said second temperature range to a temperature in said first        temperature range;    -   (c) cooling said heated suspension of (b) from a temperature in        said first temperature range to a temperature in said second        temperature range; and    -   (d) repeating steps (b) and (c) at least once,    -   thereby forming lipoprotein complexes.        91. The method of embodiment 90, wherein step (c) comprises        repeating steps (a) and (b) until at least 75%, at least 80%, at        least 85%, at least 90%, at least 95%, at least 97%, at least        98%, or at least 99% of said lipid component and/or said protein        component is in complexed form.        92. The method of embodiment 90 or embodiment 91, wherein        step (c) comprises repeating steps (a) and (b) until lipoprotein        complexes of least 75%, at least 80%, at least 85%, at least        90%, at least 95%, at least 97%, at least 98%, or at least 99%        homogeneity are obtained.        93. The method of any one of embodiments 90 to 92, wherein        step (d) comprises repeating steps (b) and (c) at least three,        at least four, or at least five times.        94. The method of any one of embodiments 90 to 93, wherein        step (c) comprises repeating steps (b) and (c) up to six, up to        eight or up to ten times.        95. The method of any one of embodiments 90 to 94, wherein,        following step (a), the suspension is maintained in the second        temperature range for at least 1, at least 2, at least 3, at        least 4 or at least 5 minutes prior to said heating step (b).        96. The method of any one of embodiments 90 to 95, wherein,        following step (a), the suspension is maintained within the        second temperature range for up to 6, up to 8, up to 10, up to        20 minutes, up to 30 minutes or up to 1 hour prior to said        heating step (b).        97. The method of any one of embodiments 90 to 96, wherein,        following step (b), the suspension is maintained within the        first temperature range for at least 1, at least 2, at least 3,        at least 4 or at least 5 minutes prior to said cooling step (c).        98. The method of any one of embodiments 90 to 97, wherein,        following step (b), the suspension is maintained within the        first temperature range for up to 6, up to 8, up to 10, up to 20        minutes, up to 30 minutes or up to 1 hour prior to said cooling        step (c).        99. The method of any one of embodiments any one of embodiments        90 to 98, wherein the resulting lipoprotein complexes are not        subject to centrifugation.        100. The method of any one of embodiments 90 to 99, wherein the        lipid component and the protein component represent the majority        of lipids and proteins and peptides, respectively, in said        starting suspension of step (a).        101. The method of any one of embodiments 90 to 100, wherein the        lipid component represents at least 60%, at least 70%, at least        80% or at least 90% of lipids in said starting suspension of        step (a).        102. The method of any one of embodiments 90 to 101, wherein the        protein component represents at least 60%, at least 70%, at        least 80% or at least 90% of proteins and peptides in said        starting suspension of step (a).        103. The method of any one of embodiments 90 to 102, wherein up        to 5%, up to 10%, up to 15% or up to 20% of lipids in said        suspension are pre-complexed to the protein component in the        starting suspension of step (a).        104. The method of any one of embodiments 90 to 103, wherein        said first temperature range includes temperatures no less than        10 degrees below and no more than 15, no more than 10, or no        more than 5 degrees above the transition temperature of said        protein component.        105. The method of any one of embodiments 90 to 104, wherein        said second temperature range includes temperatures no less than        5 or no less than 10 degrees below and no more than 5 degrees        above the transition temperature of said lipid component.        106. The method of any one of embodiments 90 to 105, wherein        said first temperature ranges spans no more than 1° C., 2° C.,        3° C., 4° C., 5° C., 7° C. or 10° C.        107. The method of any one of embodiments 90 to 106, wherein        said second temperature ranges spans no more than 1° C., 2° C.,        3° C., 4° C., 5° C., 7° C. or 10° C.        108. The method of any one of embodiments 90 to 107 further        comprising the step of forming said starting suspension.        109. The method of embodiment 108, wherein forming said        suspension comprises the step of combining a suspension of lipid        particles and a solution of said lipid-binding peptides and/or        lipid-binding proteins, each preheated at a temperature in said        first range.        110. The method of embodiment 108, wherein forming said        suspension comprises the step of mixing a population of lipid        particles and said lipid-binding peptides and/or lipid-binding        proteins pre-complexed with lipid, each preheated at a        temperature in said first range.        111. The method of embodiment 110, wherein lipid pre-complexed        with lipid-binding peptides and/or lipid-binding proteins is no        more than 5%, no more than 10%, no more than 15%, or no more        than 20% of the total lipid in said starting suspension.        112. The method of any one embodiments 109 to 111, wherein the        solution of lipids is a solution of homogenized lipids.        113. The method of embodiment 112, further comprising prior to        said combining step the step of forming a solution of        homogenized lipids using high pressure homogenization.        114. The method of embodiment 113, wherein said high pressure        homogenization is at a pressure of over 1500 bars, over 1800        bars, or over 2000 bars.        115. The method of embodiment 114, wherein said high pressure        homogenization is performed at a pressure of 1900 to 2500 bars.        116. The method of any one of embodiments 90 to 115, in which        the lipid component consists essentially of lipid particles,        said lipid particles being:    -   (i) at least 45 nm, at least 50 nm, at least 55 nm or at least        60 nm in size, as measured by DLS; and    -   (ii) up to 65 nm, up to 70 nm, up to 75 nm, up to 80 nm in size,        up to 100 nm, up to 120 nm, up to 150 nm, up to 200 nm, up to        250 nm, up to 300 nm, up to 500 nm in size as measured by DLS.        117. The method of embodiment 116, said the lipid particles        being up to 65 nm, up to 70 nm, up to 75 nm, or up to 80 nm in        size as measured by DLS.        118. The method of embodiment 116, said the lipid particles        being up to 100 nm, up to 120 nm, up to 150 nm, up to 200 nm, up        to 250 nm, up to 300 nm, up to 500 nm in size as measured by        DLS.        119. The method of any one of embodiments 90 to 116, wherein        steps (b) and (c) are repeated until lipoprotein complexes of 4        nm to 15 nm, 5 nm to 15 nm, 6 nm to 15 nm, or 8 nm to 15 nm are        obtained.        120. The method of any one of embodiments 90 to 116, wherein        steps (b) and (c) are repeated until lipoprotein complexes of 5        nm to 12 nm, 6 nm to 12 nm in size, or 8 nm to 12 nm are        obtained.        121. The method of any one of embodiments 90 to 120, in which        one, more than one or all steps are carried out under an inert        gas.        122. The method of embodiment 121, wherein the inert gas is        nitrogen.        123. The method of any one of embodiments 90 to 122, wherein        said protein component comprises or consists of lipid-binding        proteins.        124. The method of embodiment 123, wherein said lipid-binding        proteins are ApoA-1, ApoA-II, ApoA-IV, ApoC-I, ApoC-II,        ApoC-III, ApoE or mixtures thereof.        125. The method of any one of embodiments 90 to 122, wherein        said protein component comprises or consists of lipid-binding        peptides.        126. The method of embodiment 125, wherein said lipid binding        peptides are analogues of ApoA-I, ApoA-II, ApoA-IV, ApoC-I,        ApoC-II, ApoC-III, ApoE or mixtures thereof.        127. The method of any one of embodiments 90 to 126, wherein        said lipid component comprises or consists of natural lipids,        synthetic lipids, or a mixture thereof.        128. The method of embodiment 127, wherein said lipid component        comprises or consists of ether phospholipids, short chain        phospholipids, cholesterol, cholesterol derivatives,        phosphatidylcholines, phosphatidylethanolamines,        phosphatidylserines, phosphatidylinositols, sphingolipids,        phosphatidylglycerols, gangliosides, and/or cerebrosides.        129. The method of embodiment 128, wherein said lipid component        comprises or consists of egg phosphatidylcholine, soybean        phosphatidylcholine, dipalmitoylphosphatidylcholine,        dimyristoylphosphatidylcholine, distearoylphosphatidylcholine,        1-myristoyl-2-palmitoylphosphatidylcholine,        1-palmitoyl-2-myristoylphosphatidylcholine,        1-palmitoyl-2-stearoylphosphatidylcholine,        1-stearoyl-2-palmitoylphosphatidylcholine,        dioleoylphosphatidylcholine, dioleophosphatidylethanolamine,        dilauroylphosphatidylglycerol, diphosphatidylglycerol,        dimyristoylphosphatidylglycerol,        dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,        dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid,        dipalmitoylphosphatidic acid,        dimyristoylphosphatidylethanolamine,        dipalmitoylphosphatidylethanolamine,        dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine,        sphingomyelin, dipalmitoylsphingomyelin,        distearoylsphingomyelin, dipalmitoylphosphatidylglyercol salt,        acid, galactocerebroside, dilaurylphosphatidylcholine,        (1,3)-D-mannosyl(1,3)diglyceride, aminophenylglycoside, and/or        3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipids.        130. The method of any one of embodiments 90 to 126, wherein        said lipid component comprises neutral lipids.        131. The method of embodiment 130, wherein said lipid component        is predominantly neutral lipids.        132. The method of embodiment 130 or embodiment 131, wherein        said neutral lipids comprise sphingomyelins.        133. The method of embodiment 132, wherein said neutral lipids        are predominantly sphingomyelins.        134. The method of embodiment 132 or embodiment 133 in which the        sphingomyelins comprise or consist of D-erythrose-sphingomyelin        and/or D-erythrose dihydrosphingomyelin.        135. The method of any one of embodiments 130 to 135, wherein        said starting suspension further comprises negatively charged        phospholipids.        136. The method of embodiment 135, wherein said negatively        charged phospholipids comprise or consist of        phosphatidylglycerols.        137. The method of embodiment 136, wherein said        phosphatidylglycerols have C16:0 acyl chains.        138. The method of embodiment 136 or embodiment 137, wherein        said phosphatidylglycerols comprise or consist of a salt of        1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol).        139. The method of embodiment 138, wherein the salt is a sodium        salt.        140. The method of any one of embodiments 90 to 139, wherein        lipid:protein molar ratio in said starting suspension is from        about 2:1 to about 200:1.        141. The method of embodiment 140 in which the lipid:protein        molar ratio in said starting suspension is from about 10:1 to        about 125:1.        142. The method of embodiment 140 in which the lipid:protein        molar ratio in said starting suspension is from about 10:1 to        about 150:1.        143. The method of embodiment 140 in which the lipid:protein        molar ratio in said starting suspension is from about 75:1 to        125:1.        144. The method of any one of embodiments 90 to 143, wherein the        starting suspension contains negatively charged lipid, neutral        lipid and lipid-binding peptides in a molar ratio ranging from        2-6 (negatively charged lipid): 90-120 (neutral lipid): 1        (lipid-binding peptide, lipid binding protein or mixtures        thereof).        145. The method of any one of embodiments 90 to 144, wherein        said first temperature range is 55° C. to 60° C.        146. The method of any one of embodiments 90 to 145, wherein        said second temperature range is between 35° C. and 40° C.        147. The method of any one of embodiments 90 to 146, which        further comprises the step of lyophilizing the resulting        lipoprotein complexes.        148. The method of embodiment 147, further comprising the step        of adding an isotonicity agent prior to lyophilization.        149. A pharmaceutical composition comprising a population of        lipoprotein complexes, wherein said lipoprotein complexes are:    -   (a) 4 nm to 15 nm in size or 6 nm to 15 nm in size, or between 5        and 12 nm in size, or between 8 and 10 nm in size, as measured        by GPC or DLS; and    -   (b) at least 75%, at least 80%, at least 85%, at least 90%, at        least 95%, at least 97%, at least 98%, at or at least 99%        homogeneous, as reflected by a single peak in gel permeation        chromatography.        150. A method for making a pharmaceutical composition,        comprising:    -   (a) preparing a population of lipoprotein complexes according to        the method of any one of embodiments 90 to 146; and    -   (b) combining said population of lipoprotein complexes with one        or more pharmaceutically acceptable excipients.        151. The method of embodiment 150, wherein the pharmaceutical        composition is prepared under an inert gas.        152. The method of embodiment 151, wherein the inert gas is        nitrogen, helium or argon.        153. The method of any one of embodiments 150 to 152, further        comprising the step of lyophilizing the pharmaceutical        composition.        154. The method of any one of embodiments 150 to 152, further        comprising the step of aliquoting the pharmaceutical composition        into individual unit doses.        155. The method of embodiment 154, further comprising the step        of lyophilizing the individual unit doses.        156. A method for making a pharmaceutical composition,        comprising reconstituting a lyophilized preparation of        lipoprotein complexes according to the method of embodiment 147        or made by the method of embodiment 153 or embodiment 155.        157. The method of embodiment 156, further comprising combining        the reconstituted lipoprotein complexes with one or more        pharmaceutically acceptable excipients.        158. The method of embodiment 156 or embodiment 157, further        comprising the step of aliquoting the pharmaceutical composition        into individual unit doses.        159. A liquid pharmaceutical composition made by the method of        embodiment 150 or embodiment 156.        160. A lyophilized pharmaceutical composition made by the method        of embodiment 153.        161. A liquid unit dosage form made by the method of embodiment        158.        162. A liquid unit dosage form comprising a therapeutically        effective amount of the pharmaceutical composition of embodiment        149.        163. A dry unit dosage form made by the method of embodiment        155.        164. A method for treating a dyslipidemic disorder, comprising        administering to a subject in need thereof a therapeutically        effective amount of    -   (a) a lipoprotein complex according to any one of embodiments 1        to 22;    -   (b) a population of lipoprotein complexes according to any one        of embodiments 23 to 58;    -   (c) a pharmaceutical composition according to any one of        embodiments 59, 149, and 159;    -   (d) a therapeutically effective amount of lipoprotein complexes        made by the method of any one of embodiments 90 to 148;    -   (e) a unit dosage form according to embodiment 161 or embodiment        162;

(f) a lipoprotein complex that does not result in liver enzyme elevationfollowing a single administration of up to 45 mg/kg to a healthyvolunteer;

-   -   (g) a lipoprotein complex that does not result in liver enzyme        elevation following two, three, four, five or six        administrations to a human subject;    -   (h) a lipoprotein complex that does not result in liver enzyme        elevation following a single administration to a human subject        in a dose of 1 mg/kg to 20 mg/kg;    -   (i) a lipoprotein complex that does not result in liver enzyme        elevation following two, three, four, five or six        administrations to a human subject, each administration in a        dose of 1 mg/kg to 20 mg/kg;    -   (j) a lipoprotein complex that does not result in more than        two-fold triglyceride increase following a single administration        of up to 20 mg/kg to a healthy volunteer;    -   (k) a lipoprotein complex that does not result in more than        two-fold triglyceride increase following two, three, four, five        or six administrations to a human subject;

(l) a lipoprotein complex that does not result in more than two-foldtriglyceride increase following a single administration to a humansubject in a dose of 1 mg/kg to 20 mg/kg; or

-   -   (m) a lipoprotein complex that does not result in more than        two-fold triglyceride increase following two, three, four, five        or six administrations to a human subject, each administration        in a dose of 1 mg/kg to 20 mg/kg.        165. The method of embodiment 164, further comprising repeating        said administration.        166. The method of embodiment 165, wherein the administration is        repeated at an interval of 6 days to 12 days.        167. The method of embodiment 166, wherein the administration is        weekly.        168. The method of any one of embodiments 165 to 167, wherein        the administration occurs over a period of one month, five        weeks, six weeks, two months, three months, six months, one        year, 2 years, 3 years, or longer.        169. The method of any one of embodiments 165 to 167, wherein        the administration occurs once, twice, three times, four times,        five times, six times, seven times, eight times, nine times, ten        times, eleven times, or twelve times.        170. The method of any one of embodiments 164 to 169 wherein the        administration is intravenous.        171. The method of embodiment 170 wherein the administration is        by infusion.        172. The method of embodiment 171, wherein the infusion occurs        over a period of one to four hours.        173. The method of embodiment 171, wherein the infusion occurs        over a period of up to 24 hours.        174. The method of any one of embodiments 170 to 173 wherein the        amount of the lipoprotein complex ranges from about 0.25 mg/kg        ApoA-I equivalents to about 30 mg/kg ApoA-I equivalents per        administration.        175. The method of embodiment 174 wherein the amount of the        lipoprotein complex ranges from about 1 mg/kg ApoA-I equivalents        to about 15 mg/kg ApoA-1 equivalents per administration.        176. The method of embodiment 175 wherein the amount of the        lipoprotein complex ranges from about 2 mg/kg ApoA-I equivalents        to about 12 mg/kg ApoA-I equivalents per administration.        177. The method of embodiment 176 in which the amount of        lipoprotein complex is about 3 mg/kg ApoA-I equivalents per        administration.        178. The method of embodiment 176 in which the amount of        lipoprotein complex is about 6 mg/kg ApoA-I equivalents per        administration.        179. The method of embodiment 176 in which the amount of        lipoprotein complex is about 12 mg/kg ApoA-I equivalents per        administration.        180. A method for treating a dyslipidemic disorder, comprising:    -   (a) administering to a subject with an initial dose of 1 mg kg        to 12 mg/kg of:        -   (i) a lipoprotein complex according to any one of            embodiments 1 to 22;        -   (ii) a population of lipoprotein complexes according to any            one of embodiments 23 to 58;        -   (iii) a pharmaceutical composition according to any one of            embodiments 59, 149, and 159;        -   (iv) a therapeutically effective amount of lipoprotein            complexes made by the method of any one of embodiments 90 to            148;        -   (v) a unit dosage form according to embodiment 161 or            embodiment 162;        -   (vi) a lipoprotein complex that does not result in liver            enzyme elevation following a single administration of up to            45 mg/kg to a healthy volunteer;        -   (vii) a lipoprotein complex that does not result in liver            enzyme elevation following two, three, four, five or six            administrations to a human subject;        -   (viii) a lipoprotein complex that does not result in liver            enzyme elevation following a single administration to a            human subject in a dose of 1 mg/kg to 20 mg/kg;        -   (ix) a lipoprotein complex that does not result in liver            enzyme elevation following two, three, four, five or six            administrations to a human subject, each administration in a            dose of 1 mg/kg to 20 mg/kg;        -   (x) a lipoprotein complex that does not result in more than            two-fold triglyceride increase following a single            administration of up to 20 mg/kg to a healthy volunteer;        -   (xi) a lipoprotein complex that does not result in more than            two-fold triglyceride increase following two, three, four,            five or six administrations to a human subject;        -   (xii) a lipoprotein complex that does not result in more            than two-fold triglyceride increase following a single            administration to a human subject in a dose of 1 mg/kg to 20            mg/kg; or        -   (xiii) a lipoprotein complex that does not result in more            than two-fold triglyceride increase following two, three,            four, five or six administrations to a human subject, each            administration in a dose of 1 mg/kg to 20 mg/kg;    -   (b) determining whether the subject's triglyceride,        VLDL-cholesterol and/or VLDL-triglyceride is elevated to more        than two fold 4, 8, 12, 24, 48, 72, 168, 336 or 504 hours after        said administration; and    -   (c) if the subject's triglyceride, VLDL-cholesterol and/or        VLDL-triglyceride is elevated to more than two fold of the        pre-dosing levels, repeating said administration but at a lower        dose, and if the subject's triglyceride, VLDL-cholesterol and/or        VLDL-triglyceride is not elevated to more than two fold of the        pre-dosing levels, then repeating said administration at an        equivalent or greater dose.        181. The method of any one of embodiments 165 to 180, wherein        said subject has or is susceptible to hyperlipidemia or        cardiovascular.        182. The method of embodiment 181 wherein the patient is has or        is susceptible to hyperlipidemia and said hyperlipidemia is        hypercholesterolemia.        183. The method of embodiment 181 wherein the patient is has or        is susceptible to cardiovascular disease and wherein the        cardiovascular disease is atherosclerosis, stroke, myocardial        infarction, acute coronary syndrome, angina pectoris,        intermittent claudication, critical limb ischemia, atrial valve        sclerosis or restenosis.        184. The method of any one of embodiments 165 to 183 further        comprising adjunctively administering a bile-acid resin, niacin,        an anti-inflammatory agent, a statin, a fibrate, a CETP        inhibitor, a platrelt aggregation inhibitor, an anticoagulant,        an agonist of PCSK9 and/or an inhibitor of cholesterol        absorption.        185. The method of embodiment 181, further comprising        administering a statin selected from atorvastatin, rosuvastatin,        pravastatin or lovastatin.        186. The method of embodiment 181, further comprising        administering the fibrate fenofibrate.        187. The method of embodiment 181, further comprising        administering the cholesterol absorption inhibitor zetia.        188. The method of embodiment 181, further comprising        administering a CETP inhibitor selected from anacetrapib and        dalcetrapib.        189. The method of embodiment 181, further comprising        administering an antibody agonist of PCSK9 or a ligand agonist        of PCSK9.        190. The method of embodiment 181, further comprising        administering the cholesterol absorption inhibitor clopidogrel        bisulfate.        191. The method of embodiment 181, further comprising        administering the anticoagulant warfarin.        192. The method of embodiment 181, further comprising        administering the anti-inflammatory agent aspirin.        193. A composition comprising one, two or three homogeneous        populations of lipoprotein complexes.        194. The composition of embodiment 193, wherein the lipoprotein        complexes in at least one of the homogeneous populations have        the characteristics of a complex according to any one of        embodiments 1 to 22.        195. The composition of embodiment 193 or embodiment 194,        wherein at least one, two or three of said populations have the        characteristics of a population according to any one of        embodiments 23 to 58.

All cited references are incorporated herein by reference in theirentirety and for all purposes to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described are offered byway of example only, and the invention is to be limited only by theterms of the appended claims along with the full scope of equivalents towhich such claims are entitled.

1. A population of lipoprotein complexes, each comprising a lipidfraction and an apolipoprotein fraction consisting essentially of anApolipoprotein A-I (“ApoA-I”), wherein the lipoprotein complexes are atleast 85% homogeneous, as reflected by a single peak in gel permeationchromatography.
 2. The population of claim 1, further characterized byat least one of the following characteristics: (a) at least 85% of thelipoprotein complexes are in the form of particles of 4 nm to 15 nm insize as measured by dynamic light scattering (“DLS”); (b) at least 95%by weight of ApoA-I in said population is in mature form; (c) no morethan 10% by weight of ApoA-I in said population is in immature form; and(d) o more than 10% by weight of ApoA-I in the population is intruncated form.
 3. The population of claim 1, further characterized byat least one of the following characteristics: (a) no more than 5% ofeach of methionine 112 and methionine 148 in said ApoA-I in saidpopulation is oxidized; (b) no more than 4% of the amino acids of theApoA-I in said population is deamidated; (c) the population contains nomore than 1 EU of endotoxin per milligram of ApoA-I; (d) the populationcontains no more than 25 picograms host cell DNA per milligram ofApoA-I; and (e) the population contains no more than 100 nanograms hostcell protein per milligram of ApoA-I.
 4. The population of claim 1 inwhich no more than 15% by weight of the lipid in the lipid fraction insaid complexes is cholesterol.
 5. The population of claim 1 wherein saidApoA-I is a human ApoA-I protein.
 6. The population of claim 1 whereinsaid ApoA-I is a recombinant ApoA-I.
 7. The population of claim 1wherein said lipid fraction consists essentially of 95 to 99 weight %neutral phospholipid and 1 to 5 weight % negatively chargedphospholipid.
 8. The population claim 7 wherein said lipid fractionconsists essentially of 96 to 98 weight % neutral phospholipid and 2 to4 weight % negatively charged phospholipid.
 9. The population of claim1, wherein said lipid fraction consists essentially of 97 weight %neutral phospholipid and 3 weight % negatively charged phospholipid. 10.The population of claim 9, wherein the neutral lipid is naturalsphingomyelin or synthetic sphingomyelin.
 11. The population of claim 10in which the sphingomyelin is egg-sphingomyelin.
 12. The population ofclaim 9, wherein the negatively charged phospholipid comprisesphosphatidylglycerol.
 13. The population of claim 12 in which thenegatively charged phospholipid comprises a salt of1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG).
 14. Thepopulation of claim 1 which has an apolipoproteinfraction-to-phospholipid fraction ratio ranging from 1:2 to about 1:3 byweight.
 15. A pharmaceutical composition comprising the population ofclaim 1 and one or more pharmaceutically acceptable carriers, diluents,and/or excipients.
 16. The pharmaceutical composition according to claim15, in which no more than 5% of the lipoprotein is in uncomplexed form.17. The pharmaceutical composition of claim 15, in which no more than 5%of the lipid is in uncomplexed form.
 18. The pharmaceutical compositionof claim 15, comprising lipoprotein complexes having 2 ApoA-I moleculesper lipoprotein complex.
 19. The pharmaceutical composition of claim 18,comprising a lipoprotein complexes having 3 or 4 ApoA-I molecules orApoA-1 equivalents per lipoprotein complex.
 20. The pharmaceuticalcomposition of claim 15, comprising complexes in which the weight ratioof the apolipoprotein fraction to the lipid fraction is 1:2.7.
 21. Thepharmaceutical composition of claim 15 which consists essentially of thepopulation of claim 1 and one or more pharmaceutically acceptablecarriers, diluents and/or excipients.
 22. A unit dosage form comprisinga therapeutically effective amount of a pharmaceutical composition ofclaim
 21. 23. A mammalian host cell engineered to express an ApoA-1protein, said ApoA-I protein comprising an amino acid sequence having atleast 95% identity to positions 25 to 267 of SEQ ID NO:1.
 24. Amammalian cell culture comprising a plurality of the mammalian host cellaccording to claim
 23. 25. A method of producing mature, biologicallyactive ApoA-I protein, comprising culturing the mammalian host cellaccording to claim 23 under conditions in which the ApoA-I protein isexpressed and secreted.
 26. A method for preparing lipoproteincomplexes, comprising: (a) cooling a starting suspension comprising alipid component and a protein component from a temperature in a firsttemperature range to a temperature in a second temperature range,wherein said lipid component consists essentially of particles of lipidsand wherein said protein component consists essentially of lipid-bindingpeptides and/or lipid-binding proteins; (b) heating the cooledsuspension of (a) from a temperature in said second temperature range toa temperature in said first temperature range; (c) cooling said heatedsuspension of (b) from a temperature in said first temperature range toa temperature in said second temperature range; and (d) repeating steps(b) and (c) at least once, thereby forming lipoprotein complexes.
 27. Amethod for making a pharmaceutical composition, comprising: (a)preparing a population of lipoprotein complexes according to the methodof claim 26; and (b) combining said population of lipoprotein complexeswith one or more pharmaceutically acceptable excipients.
 28. A methodfor treating a dyslipidemic disorder, comprising administering to asubject in need thereof a therapeutically effective amount of; (a) apopulation of lipoprotein complexes according to claim 1; (b) alipoprotein complex that does not result in liver enzyme elevationfollowing a single administration of up to 45 mg/kg to a healthyvolunteer; (c) a lipoprotein complex that does not result in liverenzyme elevation following two, three, four, five or six administrationsto a human subject; (d) a lipoprotein complex that does not result inliver enzyme elevation following a single administration to a humansubject in a dose of 1 mg/kg to 20 mg/kg; (e) a lipoprotein complex thatdoes not result in liver enzyme elevation following two, three, four,five or six administrations to a human subject, each administration in adose of 1 mg/kg to 20 mg/kg; (f) a lipoprotein complex that does notresult in more than two-fold triglyceride increase following a singleadministration of up to 20 mg/kg to a healthy volunteer; (g) alipoprotein complex that does not result in more than two-foldtriglyceride increase following two, three, four, five or sixadministrations to a human subject; (h) a lipoprotein complex that doesnot result in more than two-fold triglyceride increase following asingle administration to a human subject in a dose of 1 mg/kg to 20mg/kg; or (i) a lipoprotein complex that does not result in more thantwo-fold triglyceride increase following two, three, four, five or sixadministrations to a human subject, each administration in a dose of 1mg/kg to 20 mg/kg.
 29. A method for treating a dyslipidemic disorder,comprising: (a) administering to a subject an initial dose of 1 mg/kg to12 mg/kg of: (i) a population of lipoprotein complexes according toclaim 1; (ii) a lipoprotein complex that does not result in liver enzymeelevation following a single administration of up to 45 mg/kg to ahealthy volunteer; (iii) a lipoprotein complex that does not result inliver enzyme elevation following two, three, four, five or sixadministrations to a human subject; (iv) a lipoprotein complex that doesnot result in liver enzyme elevation following a single administrationto a human subject in a dose of 1 mg/kg to 20 mg/kg; (v) a lipoproteincomplex that does not result in liver enzyme elevation following two,three, four, five or six administrations to a human subject, eachadministration in a dose of 1 mg/kg to 20 mg/kg; (vi) a lipoproteincomplex that does not result in more than two-fold triglyceride increasefollowing a single administration of up to 20 mg/kg to a healthyvolunteer; (vii) a lipoprotein complex that does not result in more thantwo-fold triglyceride increase following two, three, four, five or sixadministrations to a human subject; (viii) a lipoprotein complex thatdoes not result in more than two-fold triglyceride increase following asingle administration to a human subject in a dose of 1 mg/kg to 20mg/kg; or (ix) a lipoprotein complex that does not result in more thantwo-fold triglyceride increase following two, three, four, five or sixadministrations to a human subject, each administration in a dose of 1mg/kg to 20 mg/kg; (b) determining whether the subject's triglyceride,VLDL-cholesterol and/or VLDL-triglyceride is elevated to more than twofold 4, 8, 12, 24, 48, 72, 168, 336 or 504 hours after saidadministration; and (c) if the subject's triglyceride, VLDL-cholesteroland/or VLDL-triglyceride is elevated to more than two fold of thepre-dosing levels, repeating said administration but at a lower dose,and if the subject's triglyceride, VLDL-cholesterol and/orVLDL-triglyceride is not elevated to more than two fold of thepre-dosing levels, then repeating said administration at an equivalentor greater dose.
 30. The population of claim 1 in which the lipoproteincomplexes are at least 90% homogeneous.
 31. The population of claim 1 inwhich the lipoprotein complexes are at least 95% homogeneous.
 32. Thepopulation of claim 10, wherein the lipid has a peroxide value of lessthan 5 meq O/kg.